Targeted lipid particles for systemic delivery of nucleic acid molecules to leukocytes

ABSTRACT

Disclosed are targeted lipid based particles for delivery of nucleic acid molecules (such as siRNA) to leukocytes (such as T-Cells and B-cells). Further disclosed are uses of the targeted lipid based particles for treating Leukocytes-associated diseases, such as, cancer.

The Sequence Listing in ASCII text file format of 3,839 bytes in size,created on Oct. 25, 2017, with the file name “2017-10-26SequenceListing-PEER9,” filed in the U.S. Patent and Trademark Office onNov. 2, 2017, is hereby incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to targeted lipid particles for delivery ofnucleic acid molecules to Leukocytes (such as primary T lymphocytes or Blymphocytes) and uses thereof.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) can be activated by introducing synthetic shortdouble-stranded RNA fragments, termed small interfering (si)RNAs, intocells to silence genes bearing complementary sequences. RNAi can serve atool for evaluating the role of specific genes in cellular and diseaseprocesses and for therapeutic applications, by specific silencing ofdisease-relevant genes in a wide variety of diseases such as cancer,inflammation, neurodegenerative diseases and genetic disorders. However,the efficient, specific and safe delivery of RNAi payloads remains amajor challenge facing the application of RNAi therapeutics to mostdiseases.

Studies have shown significant improvements in methodologies for therecognition and delivery of siRNAs to specific disease-relevant celltypes. However, most of these delivery approaches have not beenoptimized to facilitate the intracellular trafficking of siRNAs into thecytoplasm where they provide the targeting component of the RNA-inducedsilencing complex (RISC) allowing it to direct the degradation ofspecific mRNAs.

The use of microfluidic mixing device has greatly increased the efficacyof producing Lipid-based nanoparticles (LNPs) LNPs containing mixturesof lipids, including fusogenic and ionizable amino lipids, to enhanceboth the encapsulation of siRNAs and endosomal escape once delivered tothe target cells. Recent studies have shown the efficacy of utilizingthis technology to effectively deliver siRNA to hepatocytes. Compared tomost cell types, hepatocytes have been shown to be permissive to in vivosiRNA delivery, including the delivery of naked siRNAs usinghydrodynamic injection.

Hematopoietic cells, such as leukocytes in general, and primary Tlymphocytes and B-cells, in particular, are notoriously hard totransfect with small interfering RNAs (siRNAs). Modulating immune cellsfunction, such as T cells and B-cells, by down regulating specific genesusing RNA interference (RNAi) holds tremendous potential in advancingtargeted therapies in many immune related disorders including cancer,inflammation, autoimmunity and viral infections.

CD4+ T lymphocytes play essential roles in the immune system throughtheir interaction with antigen presenting cells (APCs) and the secretionof cytokines that regulate and balance the inflammatory response.Although several strategies have been used to knockdown gene expressionin T cells in vitro and in vivo, efficient and specific delivery ofsiRNAs to T cells in therapeutically relevant doses remains a majorhurdle to the adoption of this technology for clinical applications.

MCL is an aggressive B-cell malignancy characterized by a t(11:14)chromosomal translocation that juxtaposes the proto-oncogene encodingcyclin D1 (cycD1) to the immunoglobulin heavy chain gene promoter. Thisleads to constitutive over-expression of cycD1, a protein that is notexpressed in healthy B-lymphocytes. Current MCL therapy mainly relies onconventional chemotherapy, anti-CD20 cytotoxic monoclonal antibodies,autologous stem cell transplantation, and more recently, small moleculeinhibitors of critical molecular pathways, such as the BTK inhibitor,ibrutinib. Unfortunately, relapse and progressive resistance totreatment lead to short median survival. MCL has one of the worstprognoses among lymphomas. It was previously shown that cycD1downregulation in MCL cell lines using RNAi inhibits proliferation andcauses cell cycle arrest and apoptosis. Yet, the clinical application ofthis approach is hindered by the lack of appropriate systems that coulddeliver RNAi payloads to MCL cells in an efficient and safe manner RNAitherapeutics for B-cell malignancies is especially challenging, sincethese cells are dispersed and are intrinsically resistant totransfection with nucleic acids. CD38 is expressed on the surface ofimmature hematopoietic cells, including immature B cells. Its expressionis tightly regulated during B cell ontogeny—it is expressed on bonemarrow precursors, but not mature B cells. CD38 is expressed on mostMCLs.

Several in vivo studies have suggested that LNP-based approaches may beeffective for targeting leukocytes. For example, Novobrantseva et al.demonstrated gene knockdown in murine peritoneal macrophages in vivo,and while He et. al showed robust gene silencing in human T cells invitro, and a moderate level of generalized (i.e. not specific to anylymphocyte population) silencing in spleen and bone marrow hematopoietictissues.

Nevertheless, there is a need in the art for suitable and efficient,specifically targeted delivery platforms for potent gene silencing usingsiRNA technology in leukocytes, in particular specific subsets ofleukocytes, including primary lymphocytes, such as B-cells and T-cells,and their use in new diagnostic and therapeutic approaches to dampenleukocytes related conditions, such as, inflammation and the harmfulimmune responses that occur during autoimmunity, lymphotropic viralinfections (such as HIV), and/or to treat cancers, such as, bloodcancers, including lymphomas, such as MCL.

SUMMARY OF THE INVENTION

According to some embodiments, there are provided targeted lipid-basedparticles, compositions comprising the same and uses thereof for theefficient, targeted delivery of nucleic acids (such as inhibitory RNAmolecules) to leukocytes, such as primary lymphocytes (including B-cellsand T-cells).

According to some embodiments, there are provided targeted lipid-basedparticles, which include a plurality of lipids and a PEG-maleimidemoiety/derivative, conjugated to a targeting moiety; and optionallynucleic acid encapsulated within. The targeted lipid-based particles arein particular efficient in specific delivery of nucleic acid molecules,to specific subsets of leukocytes, such as, primary lymphocytes,including T cells (such as, CD4+ T Cells) and B-cells. In someembodiments, the disclosed targeted particles can be utilized in variousdiagnostic and therapeutic applications of leukocyte-related conditions,such as, cancer, viral infections and autoimmunity.

According to some embodiments, the present invention is based in part onan advantageous composition of lipid-based particles, which enables aspecific, targeted delivery of nucleic acid molecules (such as siRNA),to leukocytes (such as lymphocytes), which is a challenging andnon-trivial task, since such cells are dispersed and are intrinsicallyresistant to transfection with nucleic acid molecules. Advantageously,in order to increase the efficacy of nucleic acid molecules (such assiRNA) delivery, the targeted tLNPs are formulated with several lipidsdesigned to improve the stability and efficacy of siRNA delivery, aswell as additional moieties, such as PEG and/or maleimide. The targetedLNPs are surface functionalized with targeting moieties (such as,antibodies, peptides or ligands) that specifically recognize cellsurface antigens whose expression is restricted to or enriched onspecific cell type, and which allow delivery to specific subtypes ofleukocytes. In some embodiments, the targeting moiety is an antibody.For example, in some exemplary embodiments, the targeted particles areconjugated to a specific anti-CD4 monoclonal antibody (mAb), whichallows specific delivery of the siRNAs encapsulated within the particlesonly to CD4+ T lymphocytes, and not other subtypes of leukocytes. Forexample, in some exemplary embodiments, the targeted particles areconjugated to a specific anti-CD38 monoclonal antibody (mAb), whichallows specific delivery of the siRNAs encapsulated within the particlesonly to B-cell lymphocytes malignancies (such as MCL), and not othersubtypes of leukocytes.

According to some embodiments, the particles disclosed herein areadvantageous as they are uniformly sized, while exhibiting a small,nanoscale mean diameter (average diameter of about 58 nm) and extremelyhigh encapsulation rate (close to 100%) of siRNA as the nucleic acid.Further, the preparation method of the particles, which utilize amicrofluidic mixer system is advantageous as it allows a temporallyefficient process, which avoids the use of extrusion of lipid particlesthrough appropriately sized filters.

According to some embodiments, the particles disclosed herein andmethods of use thereof are advantageous since knockdown of lymphaticcells, such as B cells and T-cells has not been previously demonstratedwith such lipid-based targeted particles. Likewise, it has not beenpreviously demonstrated that MCL cells can be targeted with lipid-basedparticles, in vivo, and further, that specific target gene can beknocked-down in these cells and that can in vivo selectively kill thesecells.

According to some embodiments, the particles disclosed herein areparticularly advantageous for use in hematological tissues. The bloodsupply in the hematological tissues, where MCL cells mostly reside,including spleen and bone marrow, is made up of sinusoids that allowsmall nanoparticles tissue access. Selective targeting of lymphoma cellsby antibody-targeted delivery is clinically beneficial since it canreduce the total amount of drug required for therapeutic benefit andreduce toxicity to bystander cells.

According to some embodiments, the present invention is further based onthe surprising finding that the internalization of the targetedparticles and not endosome escape is a fundamental event that takesplace as early as one hour after systemic administration, that determinetLNPs efficacy. Thus, contrast to the belief that an obstacle foreffective gene silencing by siRNA delivery systems resides in siRNAendosome escape, the findings provided herein demonstrate that theinternalization of the siRNA is a bottleneck of leukocytes targetedsiRNA delivery.

According to some embodiments, as further exemplified herein, thetargeted particles disclosed herein can be advantageously administeredsystemically (for example, by intravenous administration) to result inefficient binding and uptake into the targeted leukocytes (such as, forexample, CD4+ T lymphocytes, MCL cells), in several relevant anatomicalsites, including, for example, the spleen, inguinal lymph nodes, bloodand the bone marrow.

According to some embodiments, the particles disclosed herein comprise arobust and scalable ionizable lipid-based particles system. According tosome embodiments, the particles disclosed herein incorporate a pluralityof lipids, including fusogenic ionizable lipids (such as, DLin-MC3-DMA,DLinDMA, DLin-KC2-DMA), PEG and maleimide derivatives/moieties, and arefurther coated/conjugated to targeting antibodies (such as monoclonalantibodies) aimed at specific antigens of the desired leukocyte to betargeted (such as, for example, a T cell surface CD4 receptor, CD8receptor, CD3 receptor, CD25, CD47, CD147, CD117, CD38 (for B-cells),integrin β₇ (for B-cells), and the like) Each possibility is a separateembodiment.

According to some embodiments, the disclosed targeted particles allowsthe specific targeted delivery of nucleic acid molecules, such asinhibitory RNA molecules to a target leukocyte, to exert a downstreameffect on the specific leukocyte sub-type. In some exemplaryembodiments, the inhibitory RNA molecules can be used to induce killingof the target cell and/or modulate the fate of the cells, depending onthe target gene to be targeted by the inhibitory nucleic acid molecules.

According to some exemplary embodiments, the lipid-based particles maybe decorated with a monoclonal antibody against CD38, which is expressedon Human diseased B cells. By targeting such cells with the targetedlipid particles and by selective inhibition of a cell cycle regulator,such as, Cyclin D1, expressed on several types of the hematologicalmalignancies (such as mantle cell lymphoma, and multiple myeloma), deathof the specific cell population (and hence the malignancy) is induced.

According to some embodiments, the targeting moiety may include any typeof molecule capable of specifically recognize and interact/bind withcell surface antigens whose expression is restricted to or enriched onspecific cell. In some embodiments, the targeting moiety may be selectedfrom, but not limited to: antibodies, peptides, ligands, ligand-mimic,agonists and/or antagonists. In some embodiments, the targeting moietymay be any type of antibody, or a fragment thereof. In some embodiments,the targeting antibody is a monoclonal antibody.

According to some embodiments, there is provided a targeted particle fordelivery of a nucleic acid to leukocyte cell, the particle comprising alipid mixture comprising cationic lipid, membrane stabilizing lipid andPEG-maleimide conjugated to a targeting moiety. In some embodiments, theparticles encapsulate nucleic acid molecule.

According some embodiments, there is provided a targeted particle fordelivery of a nucleic acid to leukocyte cell, the particle comprising:a) a lipid mixture comprising cationic lipid, membrane stabilizing lipidand PEG-maleimide conjugated to a targeting moiety; and b) nucleic acidmolecules encapsulated within the particle.

In some embodiments, the particle is for targeted delivery of a nucleicacid to primary lymphocytes. In some embodiments, the lymphocytes areselected from B-cells and T-cells. In some embodiments, the lymphocytesare B-cells. In some embodiments, the lymphocytes are T-cells.

In some embodiments, the targeting moiety is configured to speciallytarget a leukocyte. In some embodiments, the targeting moiety isconfigured to specifically recognize an antigen expressed by saidleukocyte. In some embodiments, the targeting moiety is selected from anantibody, a peptide and/or a ligand. In some embodiments, the targetingmoiety comprises an antibody or a fragment thereof. In some embodiments,the antibody is anti-CD-38 antibody. In some embodiments, the targetingantibody is selected from anti-CD-antibody, anti CD8 antibody andanti-CD3 antibody. Each possibility is a separate embodiment. In someembodiments, the targeting antibody is anti-CD4 antibody. In someembodiments, the targeting antibody is anti-CD8 antibody. In someembodiments, the targeting antibody is anti-CD3 antibody.

In some embodiments, the nucleic acid comprises an interfering RNA,selected from, siRNA, miRNA, shRNA, and antisense RNA, modified formsthereof or combinations thereof.

In some embodiments, the cationic lipid may be selected from: DLinDMA,DLin-MC3-DMA, DLin-KC2-DMA, N,N-dimethyl-N′,N′-di[(9Z,12Z)-octadeca-9,12-dien-1-yl] ethane-1,2-diamine,Di-oleyl-succinyl-serinyl-tobramycin, Di-oleyl-adipyl-tobramycin,Di-oleyl-suberyl-tobramycin, Di-oleyl-sebacyl-tobramycin,Di-oleyl-dithioglycolyl-tobramycin, monocationic lipidN-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane (DOTAP), BCATO-(2R-1,2-di-O-(1′Z,9′Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate, BGSC(Bis-guanidinium-spermidine-cholesterol), BGTC(Bis-guanidinium-tren-cholesterol), CDAN (N′-cholesteryl oxycarbony1-3,7-diazanonane-1,9-diamine), CHDTAEA (Cholesterylhemidithiodiglycolyl tris(amino(ethyl)amine), DCAT(O-(1,2-di-O-(9′Z-octadecanyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate),DC-Chol (3β [N—(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol),DLKD (O,O′-Dilauryl N-lysylaspartate), DMKD (O,O′-DimyristylN-lysylaspartate), DOG (Diolcylglycerol, DOGS(Dioctadecylamidoglycylspermine), DOGSDSO(1,2-Dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine),DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine, DOSN (Dioleyl succinylethylthioneomycin), DOSP (Dioleyl succinyl paromomycin), DOST (Dioleylsuccinyl tobramycin), 1,2-Uiolcoyl-3-trimethyl ammoniopropane, DOTMA(N′[1-(2,3-Dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DPPES(Di-palmitoyl phosphatidyl ethanolamidospermine), DDAB and DODAP, or anycombination thereof. Each possibility is a separate embodiment.

In some embodiments, the cationic lipid is selected from ionizablelipids, such as, for example, DLinDMA, DLin-MC3-DMA, DLin-KC2-DMA,Di-oleyl-succinyl-serinyl-tobramycin, Di-oleyl-adipyl-tobramycin,Di-oleyl-suberyl-tobramycin, Di-oleyl-sebacyl-tobramycin,N,N-dimethyl-N′,N′-di[(9Z, 12Z)-octadeca-9,12-dien-1-yl]ethane-1,2-diamine and Di-oleyl-dithioglycolyl-tobramycin, or anycombination thereof. Each possibility is a separate embodiment.

In some embodiments, the membrane-stabilizing lipid is selected from thegroup consisting of cholesterol, phospholipids (such as,phosphatidylcholine (PC)), cephalins, sphingolipids andglycoglycerolipids

In some embodiments, the lipids may further include phosphatidylamineselected from: 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE),1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE)1,3-Dipalmitoyl-sn-glycero-2-phosphoethanolamine (1,3-DPPE)1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE),Biotin-Phosphatidylethanolamine,1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine(DMPE),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), andDipalmitoylphosphatidylethanolamine (DPPE).

In some embodiments, the particle further include one or more PEGderivatives. In some embodiments, the additional PEG derivative isselected from: DMG-PEG, PEG-cDMA, 3-N-(-methoxy poly(ethyleneglycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine; PEG-cDSA,3-N-(-methoxy poly(ethyleneglycol)2000)carbamoyl-1,2-distearyloxy-propylamine, PEG-Amine, DSPE-PEG,or combinations thereof.

In some embodiments, the maleimide moiety is conjugated to a PEGderivative.

In some embodiments, the lipid mixture comprises DLin-MC3-DMA,cholesterol, DSPC, PEG-DMG and DSPE-PEG-maleimide. In some embodiments,the lipid mixture comprises DLinDMA, cholesterol, DSPC, PEG-DMG andDSPE-PEG-maleimide. In some embodiments, the lipid mixture comprisesDLinKC2DMA, cholesterol, DSPC, PEG-DMG and DSPE-PEG-maleimide.

In some embodiments, the percentage of encapsulation of the nucleic acidis over 90%.

In some embodiments, there is provided a composition comprising aplurality of the targeted particles. In some embodiments, there isprovided a composition comprising a plurality of the targeted particlesencapsulating nucleic acid molecule(s). In some embodiments, there isprovided a pharmaceutical composition comprising the plurality ofparticles in a dosage form suitable for administration via a routeselected from oral and parenteral. In some embodiments, theadministration is systemic.

According to some embodiments, there is provided a method for treating aleukocyte associated disease, comprising the step of administering to asubject in need thereof a pharmaceutical composition comprising thetargeted particles and suitable nucleic acid molecules encapsulatedtherein. In some embodiments, the leukocyte associated disease iscancer. In some embodiments, the leukocyte associated disease is bloodcancer. In some embodiments, the blood cancer is selected from lymphoma,leukemia and multiple myeloma. In some embodiments, the cancer islymphoma. In some embodiments, the lymphoma is selected from B-celllymphoma and T-cell lymphoma. In some embodiments, the B-cell lymphomais selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma. In someembodiments, the B-cell lymphma is selected from Burkitt lymphoma,chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL),diffuse large B-cell lymphoma, follicular lymphoma, marginal-zone B-celllymphoma, Nodal marginal zone B cell lymphoma (NMZL), Splenic marginalzone lymphoma (SMZL), Intravascular large B-cell lymphoma, Primaryeffusion lymphoma, Lymphomatoid granulomatosis, Primary central nervoussystem lymphoma, ALK-positive large B-cell lymphoma, Plasmablasticlymphoma and mantle cell lymphoma (MCL). In some embodiments, thelymphoma is Mantle cell lymphoma (MCL). In some embodiments, the T-celllymphoma is selected from: Peripheral T-cell lymphoma, Anaplastic largecell lymphoma, Angioimmunoblastic Lymphoma, Cutaneous T-cell lymphoma,Adult T-cell Leukemia/Lymphoma (ATLL), Blastic NK-cell Lymphoma,Enteropathy-type T-cell lymphoma, Hematosplenic gamma-delta T-cellLymphoma, Lymphoblastic Lymphoma, Nasal NK/T-cell Lymphomas andTreatment-related T-cell lymphomas. In some embodiments, for treatingthe leukocyte associated disease, the siRNA encapsulated within theparticles is siRNA against a cell cycle regulator. In some embodiments,the cell cycle regulator may be selected from: Polo-like Kinase 1 (PLK),Cyclin D1, CHK1, Notch pathway genes, PDGFRA, EGFRvIII, PD-L1, RelB,STAT1, STAT3, MCL1, CKAP5, RRM1, SF3A1 and CDK11B, and the like, orcombinations thereof. In some embodiments, the cell cycle regulator isCyclin D1 (CycD1).

According to some embodiments, there is provided use of a pharmaceuticalcomposition for treating a leukocyte-associated disease, the compositioncomprising: a) targeted particles for delivery of a nucleic acid to aleukocyte, the particle comprising a lipid mixture comprising cationiclipid, membrane stabilizing lipid and PEG-maleimide conjugated to atargeting moiety; and b) nucleic acid encapsulated within the particle.In some embodiments, the pharmaceutical compositions comprises aplurality of particles.

According to some embodiments, there is provided use of a pharmaceuticalcomposition for treating a leukocyte-associated disease, the compositioncomprising targeted particle for delivery of a nucleic acid to aleukocyte, the particle comprising: a) a lipid mixture comprisingcationic lipid, membrane stabilizing lipid and PEG-maleimide conjugatedto a targeting moiety; and b) nucleic acid encapsulated within theparticle.

In some embodiments, the leukocyte-associated disease is cancer. In someembodiments, the cancer is lymphoma. In some embodiments, the lymphomais Mantle cell lymphoma (MCL).

In some embodiments, the nucleic acid is siRNA. In some embodiments, thesiRNA is directed against a cell cycle regulator. In some embodiments,the cell cycle regulator is Cyclin D1.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thefigures and by study of the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. Dimensionsof components and features shown in the figures are generally chosen forconvenience and clarity of presentation and are not necessarily shown toscale. The figures are listed below.

FIG. 1A—Schematic illustration of the preparation of targetedlipid-based particles (LNPs);

FIG. 1B—Schematic illustration of the preparation of targetedlipid-based particles (LNPs) conjugated to an anti-CD-38 targetingantibody (specifically directed to MCL cells);

FIG. 2A—Transmission electron microscopy (TEM) images of unconjugatedLNPs (lipid based particles not conjugated to a targeting antibody),(left-hand panel) or antibody-targeted lipid-based particles (tLNPs)(right-hand panel);

FIG. 2B—Transmission electron microscopy image of αCD38-LNPs-siRNA.White scale bar: 100 nm.

FIG. 3—Images of Dot blot analysis for antibody presence on the surfaceof isoLNPs (isotype-control mAb), tLNPs (targeted particles) or LNPs (noantibody) as a control;

FIGS. 4A-D show tLNPs binding and internalization into CD4⁺ T cellsex-vivo. Primary splenocytes were incubated with tLNPs (siCy5) orisoLNPs (siCy5) as a control for 30 min. CD4⁺ cells were then labeledwith anti-CD4 PE. FIG. 4A—Flow cytometry dot blot analysis of gated livelymphocyte population, dots indicating tLNPs (upper right quartet) andisoLNPs (upper left quartet); FIG. 4B—Corresponding histograms of % GMFIcalculated for CD4⁺ population over CD4⁻ populations including CD8 andCD19 cells, data presented as mean±SD, n=3, ***p<0.0005; FIG.4C—Splenocytes were incubated for further 30 min at 37° C. to allowinternalization. Cells were stained with Hoechst and calcein for nuclearand cytoplasm detection followed by membrane staining withanti-CD4-AF594. Cells were analyzed by confocal microscopy forinternalization (left panel); representative individual images of area(a) shown in the right panels. GMFI: Geometric mean fluorescenceintensity; FIG. 4D—Ex vivo specific binding of tLNPs: Splenocytes werecollected from mouse and lymphocytes were incubated with tLNPs (siCy5)for 30 min at 4° C., followed by 30 min at room temperature. Cells werewashed and stained with anti-CD4-PE (originally red) and anti-CD8-FITC(originally green), then analyzed by confocal microscopy. Merged image(left panel), individual images of each fluorophore (right). tLNPs shown(originally cyan);

FIGS. 5A-E show tLNPs can target blood circulating CD4⁺ T cells in vivoand induce gene silencing. FIG. 5A—Targeting blood CD4⁺ T cells invivo—One hour post i.v. administration of tLNPs (siCy5) or isoLNPs(siCy5), circulating lymphocytes were isolated and stained with a set ofantibodies (anti-CD4 PE, anti-CD3 PerCp and anti-CD8 FITC).Representative histograms of flow cytometry analysis of LNPs bindingprofile in blood lymphocytes from two independent experiments are shown;FIG. 5B—Corresponding bar graphs of the histrograms of FIG. 5A arepresented as percent GMFI values over mock, data represent mean±SD, n=3,*p<0.05; FIG. 5C—tLNPs silence CD45 in blood T lymphocytes. Mice wereinjected with tLNPs (siCD45), saline (mock), tLNPs(siLuc),isoLNPs(siCD45) or LNPs (siCD45) as controls. Five days post i.v.administration, circulating lymphocytes were isolated and stained forCD45 expression. The flow cytometry analysis of dot blots gated for livelymphocytes is shown. Results were mean of two independent experiments,n=5; FIG. 5D—Corresponding histograms of FIG. 5C, of percent CD45silencing calculated from CD4 gated populations, data represent mean±SD,n=5, **p<0.005; FIG. 5E—CD45 is silenced specifically in CD4⁺circulating T cells. Five days after administration of saline or tLNPs(siCD45), circulating lymphocytes were isolated and stained with a setof antibodies. Presented is a Flow cytometry dot blot profile of CD45silenced cell population gated for CD4⁺/CD3⁺ cells, n=5; KD—knock down;

FIG. 6—In vivo immune activation study. Serum was collected from miceinjected with tLNPs (siCD45) or LPS and analyzed for pro inflammatorycytokines by ELISA. Bar graphs show the concentration (pg/ml) of IL-10,TNF-alpha, and IL-17 as determined by ELISA;

FIG. 7—Bio-distribution of tLNPs and isoLNPs: One hour postadministration of tLNPs (siCy5) or isoLNPs (siCy5), lymphocytes wereisolated from spleen, blood, lymph and bone marrow. Cell were stainedfor anti-CD4-PE and analyzed by flow cytometry. Represent histograms ofpercent GMFI values for Cy5 calculated form CD4 gated populationsnormalized to mock. Error bars represents mean±SD, n=3, NS-notsignificant, *p<0.05, ***p<0.0005;

FIGS. 8A-D—tLNPs target and silence CD4⁺ T cells in hematopoieticorgans. FIG. 8A—tLNPs binding CD4⁺ cells in diverse hematopoietic organsin vivo. One-hour post administration of siCy5 containing tLNPs(originally range) or saline (originally gray), spleen, lymph nodes,bone marrow and blood lymphocytes were isolated and stained with a setof antibodies (anti-CD4 PE, anti-CD3 PerCp and anti-CD8 FITC).Representative dot blot analysis for gated live lymphocytes ispresented, data were obtained from two independent experiments, n=5mice/group; FIG. 8B—CD4 specific silencing in hematopoietic organs. Fivedays after administration of tLNPs (siCD45) (orange) or saline (gray)administration, spleen, lymph nodes, bone marrow and blood lymphocyteswere isolated and incubated with a set of antibodies (anti-CD45 AF647,anti-CD4 PE, anti-CD3 PerCp and anti-CD8 FITC). Representative dot blotanalysis for gated live CD4⁺ lymphocytes; FIG. 8C—Corresponding bargraphs of FIG. 8B, error bars represent mean±SD, n=5 mice/group,***p<0.0005, **p<0.005 are compared to mock treated sample; FIG. 8D—TheSilencing of CD45 in CD4⁺ T cells at the mRNA level. Gated CD45^(KD) andmock CD4⁺ T cells were collected by BD FACSARIAIII™ cell sorter, mRNAwas isolated and CD45 mRNA levels were tested by qPCR. All values arenormalized to murine PPIB gene expression (endogenous control);

FIG. 9—tLNPs inducing gene silencing in CD4+ cells specifically inhematopoietic organs. Five days after tLNPs(siCD45) administration,spleen, lymph and blood lymphocytes were isolated and incubated with aset of antibodies (anti-CD45 APC, anti-CD4 PE, anti-CD3 PerCp andanti-CD8 FITC). Representative dot blot analysis for gated live CD4+,CD8+ or CD3− lymphocytes is presented. Silenced cells (originallyorange) and unsilenced cells (originally gray) are shown;

FIGS. 10A-B—Dose dependent bio-distribution and silencing. FIG. 10A-Lymphocytes from spleen, blood, lymph and bone marrow were isolated, onehour post administration of tLNPs (siCy5) at 1 mg/kg or 2 mg/kg siRNAdose. Representative histograms of percent GMFI values for Cy5calculated from gated CD4 populations normalized to mock; FIG. 10B- Fivedays after the administration of tLNPs (siCD45) at 0.5, 1 and 2 mg/kgsiRNA amounts, lymphocytes were collected from different organs, stainedand analyzed by flow cytometry. Representative histograms for CD45silenced CD4 cells (%) from gated CD4+/CD45+ populations. Error barsrepresents mean±SD, n=3, *p<0.05, ***p<0.0005;

FIGS. 11A-B: CD4low T cells are positive for CD3. One hour after tLNPs(siCD45) administration, splenocytes were isolated and incubated withanti-CD4 PE and anti-CD3 PerCp. FIG. 11A—Flow cytometry dot blotanalysis presenting CD4high, CD4low and CD4− populations; FIG.11B—Corresponding histograms representing high staining of CD3 for bothCD4low and CD4high populations;

FIGS. 12A-E: LNPs internalization by CD4 subset followed by functionalsilencing. FIG. 12A- one hour post tLNPs (siCy5) administration,splenocytes were isolated, stained with anti-Rat Fc followed by anti-CD4PE and analyzed by flow cytometry, analysis is presented on gatedpopulations; FIG. 12B- Splenocytes collected from tLNPs (siCy5) treatedmice were stained with Hoechst, calcein and anti-CD4 PE. Cells wereanalyzed by confocal microscopy. CD4high and CD4low cells are markedwith green and white arrows respectively; FIG. 12C—One hour postadministrations of tLNPs or iso LNPs (siCD45), splenocytes werecollected, labeled with anti-CD4 PE, CD4+ T cells of isoLNPs, CD4high(green), CD4low (red) cells of tLNPs were separated using BDFACSArieIII™ cell sorter; FIG. 12D—Sorted cells were cultured in vitrofor 3 days, then stained with anti-CD45- AF647 and analyzed by flowcytometry; FIG. 12E- Corresponding bar graphs, error bars represent mean±SD, n=3 mice/group, *p=0.006;

FIGS. 13A-B: Bio distribution of tLNPs. Mice were injected tLNPs (siCy5)and splenocytes were isolated at 1 h and 4 h time points, stained withanti-CD4-PE and analyzed by flow cytometry. FIG. 13A—The distribution oftLNPs in mouse at 1 h and 4 h plotted against GMFI values of Cy5calculated from gated CD4 population; FIG. 13B—Distribution of tLNPs inCD4low cells after 1 h and 4 h administration of tLNPs. Error barsrepresents mean±SD, n=3, NS=not significant;

FIGS. 14A-B—MCL-xenograft model. Granta-GFP cells were i.v. injectedinto CB-17 SCID mice. FIG. 14A—Indicated organs were extracted when micedeveloped hind-leg paralysis. Single-cell suspensions were prepared andanalyzed for Granta-GFP presence by flow cytometry (GFP+/hCD45+). FIG.14B—H&E stain on femur bones from untreated and MCL-bearing mice.

FIGS. 15A-D—αCD38 mAb targets MCL cells and induces cellularinternalization. FIG. 15A—αCD38 mABb binding to 4 MCL cell lines.Continuous line: αCD38. Dashed lines: isotype control. FIG. 15B—αCD38mAb binding to MCL cells (CD5+/CD19+) relative to non-MCL leukocytespresent in blood samples from MCL patients. Each dot represent onesample (n=5), horizontal bar represents mean (*P<0.05; two-tailedStudent's t test for paired values). FIG. 15C—αCD38 mAb internalizationupon binding to Granta-519 cells. White scale bar: 20 μm. FIG. 15D—invivo binding of αCD38 mAb to different organs in representative MCLbearing mouse. Indicated organs were extracted 2 h post αCD38 mAbinjection, suspended and analyzed by flow cytometry. αCD38 mAb can befound on most MCL cells (Granta-GFP cells) in the BM.

FIGS. 16A-C. αCD38-LNPs-siRNA mediate active delivery of siRNAspecifically into MCL cells. FIG. 16A—Granta-GFP (left) or Jeko-GFP(right) were co-cultured with TK-1 (murine T-lymphoma) cells andincubated with αCD38-LNPs-siRNA entrapping labeled siRNA. FIG.16B—Mononuclear cells from 2 blood samples of MCL patients wereincubated with αCD38-LNPs-siRNA including labeled siRNA. FIG. 16A andFIG. 16B exhibit siRNA-LNPs binding to non-B cells (originally grey),MCL cells (originally red) or MCL cells in samples incubated with freecompeting αCD38 mAbs prior to αCD38-LNPs-siRNA incubation (originallypurple). FIG. 16C—Granta-519 cells uptake of siRNA delivered viaindicated LNPs and visualized by live confocal microscopy. White scalebar: 20 μm.

FIGS. 17A-E—αCD38-LNPs-siRNA mediate active delivery of siRNAspecifically into MCL cells and induce anti-tumor gene silencing. FIG.17A and FIG. 17B- Granta-519 (higher panels) or Jeko-1 (lower panels)were incubated with mock (black), αCD38-LNPs-siLuc (grey) orαCD38-LNPs-siCycD1 (red). 48h post treatment, cells were analyzed forcycD1 protein expression by flow cytometry. FIG. 17A show representativedata from one of five (Granta) or three (Jeko) experiments. Continuouslines: cycD1 staining. Dashed lines: isotype control. Filled histogram:unstained. Complete data are represented in FIG. 17B- Bar plotsrepresent mean±SEM of cycD1 expression normalized to mock (**P<0.01;***P<0.001; One-way ANOVA test with Bonferroni correction). FIG. 17C-qRT-PCR quantification of cyclin D1 transcripts in MCL cell lines. RNAwas extracted from cells 48 h hours following treatment with mock,αCD38-LNPs-siLuc or αCD38-LNPs-siCycD1. Bar plots represent mean±SEM ofcycD1 expression relative to mock (n=3 independent experiments per cellline, **P<0.01; One-way ANOVA test with Bonferroni correction); FIG.17D—Cell cycle distribution of cells 48 h post treatments with mock(black), αCD38-LNPs-siLuc (grey) or αCD38-LNPs-siCycD1 (originally red)analyzed by flow cytometry. Bars represent mean percentage±SEM of n=4from two independent experiments per cell line (**P<0.01; ***P<0.001;

P<10-4; One-way ANOVA test with Bonferroni correction); FIG. 17E-qRT-PCR analysis of CCND1, CCND2 and CCND3 mRNA levels 24, 48, 72 and 96hours post electroporation in Granta-519 (left) and Jeko-1 (right)cells. Expression was normalized to both eIF3a and eIF3c genes anddepicted as mRNA concentration relative to cells electroporated withsiLuc. Data are mean±SEM of three independent experiments;

FIGS. 18A-D. αCD38-LNPs-siCycD1 target MCL xenografts in the BM andprolong the survival of diseased mice. FIG. 18A FIG. 18B—Mice bearinghuman MCL cells were i.v. injected with mock, isotype- orαCD38-LNPs-siRNAs. Bone marrow cells were extracted 2 h later andanalyzed for LNPs binding as detected by siRNA fluorescence via flowcytometry. Human MCL (left) and murine (right) cells were gatedseparately based on GFP, hCD20 and mCD45 expression. Cells with siRNAfluorescence levels higher than in top 1% cells from mock-treated mousewere considered positive (siRNA-positive cells are colored, cut-offrepresented by vertical bar). In FIG. 18A, shown are dot plots for onerepresentative animal from each treatment group (isotype—n=2; αCD38n=3). Number indicates percentage of siRNA-positive cells. Completeresults are shown in FIG. 18B. Bar plots represent mean±SEM (ns P>0.05;***P<0.001; two-tailed Student's t test). FIG. 18C—Survival curves ofMCL bearing mice. Corresponding treatments (1 mg siRNA/kg body) wereadministrated at 9 time-points (arrows) via retro-orbital route. n=10animals per group. P values and significance were determined by Log-rankMantel-Cox test with Bonferroni correction (*P<0.05); FIG. 18D—Repeatedi.v. administration of αCD38-LNPs-siRNA did not affect mice bodyweightAnimals were inoculated and treated as in FIG. 18C. Shown are meanweight±SEM of mice (n=10 per group).

DETAILED DESCRIPTION

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below. It is to be understood that theseterms and phrases are for the purpose of description and not oflimitation, such that the terminology or phraseology of the presentspecification is to be interpreted by the skilled artisan in light ofthe teachings and guidance presented herein, in combination with theknowledge of one of ordinary skill in the art.

As referred to herein, the terms “nucleic acid”, “nucleic acidmolecules” “oligonucleotide”, “polynucleotide”, and “nucleotide” mayinterchangeably be used herein. The terms are directed to polymers ofdeoxyribonucleotides (DNA), ribonucleotides (RNA), and modified formsthereof in the form of a separate fragment or as a component of a largerconstruct, linear or branched, single stranded, double stranded, triplestranded, or hybrids thereof. The term also encompasses RNA/DNA hybrids.The polynucleotides may include sense and antisense oligonucleotide orpolynucleotide sequences of DNA or RNA. The DNA or RNA molecules may be,for example, but not limited to: complementary DNA (cDNA), genomic DNA,synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA moleculesuch as, for example, mRNA, shRNA, siRNA, miRNA, Antisense RNA, and thelike. Each possibility is a separate embodiment. The terms furtherinclude oligonucleotides composed of naturally occurring bases, sugars,and covalent internucleoside linkages, as well as oligonucleotideshaving non-naturally occurring portions, which function similarly torespective naturally occurring portions.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “antigen” as used herein refers to a molecule or a portion of amolecule capable of being specifically bound by an antibody. An antigenmay have one or more epitopes. In some embodiments, the antigen is aprotein specifically expressed by a specific cell. In some embodiments,the antigen is a membranous protein. In some embodiments, the antigen isexpressed on the exterior membrane of a cell. In some embodiments, theantigen is a cell surface protein. In some embodiments, the antigen is acell surface receptor.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (mAb), including full length or intact monoclonalantibodies, polyclonal antibodies, multivalent antibodies,multi-specific antibodies (e.g., bi-specific antibodies), and antibodyfragments long enough to exhibit the desired binding/recognizingactivity. An “Antibody fragments” comprise a portion of an intactantibody, generally including an antigen binding site of the intactantibody and thus retaining the ability to bind an antigen.

The term “targeting moiety” is directed to any type of molecule capableof specifically recognizing and interact/bind with cell surface antigenswhose expression is restricted to or enriched on specific cell. In someembodiments, the targeting moiety is selected from, but not limited to:antibodies, peptides, ligands, ligand-mimic, agonists and/orantagonists. In some embodiments, the targeting moiety may be any typeof antibody, or a fragment thereof. In some embodiments, the targetingantibody is a monoclonal antibody.

The term “construct”, as used herein, refers to an artificiallyassembled or isolated nucleic acid molecule which may include one ormore nucleic acid sequences, wherein the nucleic acid sequences mayinclude coding sequences (that is, sequence which encodes an endproduct), regulatory sequences, non-coding sequences, or any combinationthereof. The term construct includes, for example, vector but should notbe seen as being limited thereto.

“Expression vector” refers to constructs that have the ability toincorporate and express heterologous nucleic acid fragments (such as,for example, DNA), in a foreign cell. In other words, an expressionvector comprises nucleic acid sequences/fragments (such as DNA, mRNA,tRNA, rRNA), capable of being transcribed. Many prokaryotic andeukaryotic expression vectors are known and/or commercially available.Selection of appropriate expression vectors is within the knowledge ofthose having skill in the art. In some exemplary embodiments, theexpression vector may encode for a double stranded RNA molecule in thetarget site.

The term “expression”, as used herein, refers to the production of adesired end-product molecule in a target cell. The end-product moleculemay include, for example an RNA molecule; a peptide or a protein; andthe like; or combinations thereof.

As used herein, the terms “introducing” and “transfection” mayinterchangeably be used and refer to the transfer of molecules, such as,for example, nucleic acids, polynucleotide molecules, vectors, and thelike into a target cell(s), and more specifically into the interior of amembrane-enclosed space of a target cell(s). The molecules can be“introduced” into the target cell(s) by any means known to those ofskill in the art, for example as taught by Sambrook et al. MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork (2001), the contents of which are incorporated by reference herein.Means of “introducing” molecules into a cell include, for example, butare not limited to: heat shock, calcium phosphate transfection, PEItransfection, electroporation, lipofection, transfection reagent(s),viral-mediated transfer, and the like, or combinations thereof. Thetransfection of the cell may be performed on any type of cell, of anyorigin, such as, for example, human cells, animal cells, plant cells,virus cell, and the like. The cells may be selected from isolated cells,tissue cultured cells, cell lines, cells present within an organismbody, and the like.

As referred to herein, the term “target site” refers to the location inwhich the nucleic acid is directed to and/or the site in which thenucleic acid is to exert its biological effect. In some exemplaryembodiments, the target site is a cell that may be selected from, butnot limited to: a culture cell (primary cell or cell-line derived cell),and a cell within an organism body; a tissue, an organ, a microorganism(such as, for example, virus, bacteria, parasite), and the like. Theorganism may be any organism, such as, but not limited to: a mammal,such as human or an animal, an animal which is not a mammal (such as,for example, avian, Fish, and the like), and the like. In some exemplaryembodiments, the target site is a subcellular location or cellularorganelle (such as, for example, nucleus, cytoplasm, and the like). Insome embodiments, the target site is a leukocyte, or a subset thereof.In some embodiments, the target site is a specific T-cell. In someembodiments, the target site is a specific B-cell. In some embodiments,the target site is a lymphoma.

The term “treating” and “treatment” as used herein refers to abrogating,inhibiting, slowing or reversing the progression of a disease orcondition, ameliorating clinical symptoms of a disease or condition orpreventing the appearance of clinical symptoms of a disease orcondition. The term “preventing” is defined herein as barring a subjectfrom acquiring a disorder or disease or condition.

The term “treatment of cancer” is directed to include one or more of thefollowing: a decrease in the rate of growth of the cancer (i.e. thecancer still grows but at a slower rate); cessation of growth of thecancerous growth, i.e., stasis of the tumor growth, and, the tumordiminishes or is reduced in size. The term also includes reduction inthe number of metastases, reduction in the number of new metastasesformed, slowing of the progression of cancer from one stage to the otherand a decrease in the angiogenesis induced by the cancer. In mostpreferred cases, the tumor is totally eliminated. Additionally includedin this term is lengthening of the survival period of the subjectundergoing treatment, lengthening the time of diseases progression,tumor regression, and the like. In some embodiments, the cancer is ablood cancer.

The term “Leukocytes” is directed to white blood cells (WBCs), producedand derived from a multipotent, hematopoietic stem cell in the bonemarrow. The white blood cells have nuclei, and types of white bloodcells can be classified in into five main types, including, neutrophils,eosinophils, basophils, lymphocytes, and monocytes, based on functionalor physical characteristics. The main types may be classified intosubtypes. For example, lymphocytes include B cells, T cells, and NKcells. B-cells, for example, release antibodies and assist activation ofT cells. T cells, for example, can be classified to several subtypes,including: T-helper cells (CD4+ Th) which activate and regulate T and Bcells; cytotoxic T cells (CD8+) that can target and kill virus-infectedcells and tumor cells; Gamma-delta T cells (γδ T cells) which can bridgebetween innate and adaptive immune responses and be involved inphagocytosis; and Regulatory (suppressor) T cells which modulate theimmune system, maintain tolerance to self-antigens, and abrogateautoimmune conditions.

As used herein, the term “LNP” is directed to lipid-based nanoparticles.The lipid based particles may be targeted, whenconjugated/attached/associated with a targeting moiety, such as, anantibody. In some embodiments, the particles are nano-particles. In someembodiments, the term “tLNP” is directed to the lipids based particleswhich are attached/coated/conjugated/linked to a targeting moiety. Insome embodiments, the LNP encapsulates a nucleic acid.

The terms “maleimide” and “maleimide moiety” may interchangeably be usedand are directed to a chemical compound having the formula H₂C₂(CO)₂NH.In some embodiments, the maleimide may be bound/conjugated/linked toanother derivative, such as, PEG derivative. In some embodiments, amaleimide moiety may include two maleimide groups connected by thenitrogen atoms (Bismaleimides). In some embodiments, stablecarbon-sulfur bond can be formed between the maleimide (the double bondthereof) and thiol group(s).

The term “plurality” as used herein is directed to include more than onecomponent.

As used herein, the term “about” refers to +/−10%.

According to some embodiments of the present invention, there isprovided a targeted lipid particle for delivery of a nucleic acid to aleukocyte, which comprises a lipid phase (membrane/mixture) comprising aplurality of lipids (including cationic lipid(s), membrane stabilizinglipid(s) and optionally additional lipids, such as, but limited to,ionized lipids and/or phosphatidylethanolamine(s)), one or morePEG-Amine derivatives and maleimide, conjugated to a targeting moiety.In some embodiments, the particles further include (encapsulate) anucleic acid. In some embodiments, the maleimide is conjugated to alipid, via PEG derivative. In some embodiments, the disclosed particlesare used as an efficient delivery system (both in-vitro and in-vivo) todeliver the nucleic acid molecule to a target leukocyte, such as, alymphocyte including B-cells and T-cells primary lymphocytes. The targetsite may be an in-vivo or in-vitro target site.

According to some embodiments, there is provided a cationic particle fordelivery of a nucleic acid, comprising: a lipid membrane/mixturecomprising a cationic lipid, a membrane stabilizing lipid and maleimideconjugated to a targeting moiety; and a nucleic acidencapsulated/carried within the particles. In some embodiments, thetargeting moiety is at least partially coating the external surface ofthe particles. In some embodiments, the targeting moiety is configuredto recognize, target and/or bind a specific antigen or epitope of thetarget leukocyte, thereby allowing targeting of the particle to thetarget cell.

According to additional embodiments, the present invention provides acomposition comprising a plurality of particles, the particlescomprising a lipid phase/membrane/mixture comprising a plurality oflipids comprising at least a cationic lipid, a membrane stabilizinglipid and at maleimide derivative, the maleimide derivative isconjugated to a targeting moiety (such as a targeting antibody); andfurther encapsulate/carry a nucleic acid within the particles. In someembodiments, the particles further include one or more PEG-derivatives.

Reference is now made to FIG. 1A, which is a schematic illustration ofpreparation of targeted particles, according to some embodiments. Asshown, a mixture of lipids, including maleimide moieties/derivatives ismixed with nucleic acid molecules (exemplary shown as siRNA molecules).After formation of the particles (by mixing in a microfluidic mixer), atargeting moiety (shown as a targeting antibody, which is reduced, forexample by thilation with DTT), is conjugated to the maleimide moiety,to thereby form the targeted particles.

Reference is now made to FIG. 1B, which is a schematic illustration ofpreparation of targeted lipid-based particles (LNPs) conjugated to ananti-CD-38 targeting antibody (for targeting, for example, MCL cells),according to some embodiments. As shown, a mixture of lipids, includingmaleimide moieties/derivatives is mixed with nucleic acid molecules(exemplary shown as siRNA molecules). After formation of the particles(by mixing in a microfluidic mixer), a targeting antibody (anti-CD38monoclonal antibody), is reduced (for example by thilation with DTT).The reduced anti-cD38 monoclonal antibody is attached/conjugated to themaleimide moiety, to form the targeted particles. Thereafter, unboundantibodies are removed (for example, by gel-filtration chromatography),to result with the purified anti-CD38 targeted lipids based particles(tLNPs).

According to some exemplary embodiments, the plurality of lipids of thelipid particles may be of natural or synthetic source and may beselected from, but not limited to: cationic lipids,phosphatidylethanolamines, ionized lipids, membrane stabilizing lipids,phospholipids, and the like, or combinations thereof. Each possibilityis a separate embodiment.

In some embodiments, the cationic lipid may be selected from ionizablelipid or permanently charged amine cationic lipids.

In some embodiments, the cationic lipids may be synthetic cationiclipids.

In some embodiments, the cationic lipids may be selected from: DLinDMA,DLin-MC3-DMA, DLin-KC2-DMA, Di-oleyl-succinyl-serinyl-tobramycin,Di-oleyl-adipyl-tobramycin, Di-oleyl-suberyl-tobramycin,Di-oleyl-sebacyl-tobramycin, N,N-dimethyl-N′,N′-di[(9Z,12Z)-octadeca-9,12-dien-1-yl] ethane-1,2-diamine,Di-oleyl-dithioglycolyl-tobramycin, monocationic lipidN-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane (DOTAP), BCATO-(2R-1,2-di-O-(1′Z,9′Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate, BGSC(Bis-guanidinium-spermidine-cholesterol), BGTC(Bis-guanidinium-tren-cholesterol), CDAN (N′-cholesteryl oxycarbony1-3,7-diazanonane-1,9-diamine), CHDTAEA (Cholesterylhemidithiodiglycolyl tris(amino(ethyl)amine), DCAT(O-(1,2-di-O-(9′Z-octadecanyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate),DC-Chol (3β [N—(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol),DLKD (O,O′-Dilauryl N-lysylaspartate), DMKD (O,O′-DimyristylN-lysylaspartate), DOG (Diolcylglycerol,DOGS(Dioctadecylamidoglycylspermine), DOGSDSO(1,2-Dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine),DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine, DOSN (Dioleyl succinylethylthioneomycin), DOSP (Dioleyl succinyl paromomycin), DOST (Dioleylsuccinyl tobramycin), 1,2-Uiolcoyl-3-trimethyl ammoniopropane, DOTMA(N′[1-(2,3-Dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DPPES(Di-palmitoyl phosphatidyl ethanolamidospermine), DDAB and DODAP, or anycombination thereof. Each possibility is a separate embodiment.

In some exemplary embodiments, the cationic lipid may be selected from:DLinDMA, DLin-MC3-DMA, DLin-KC2-DMA,Di-oleyl-succinyl-serinyl-tobramycin, Di-oleyl-adipyl-tobramycin,Di-oleyl-suberyl-tobramycin, N,N-dimethyl-N′,N′-di[(9Z,12Z)-octadeca-9,12-dien-1-yl] ethane-1,2-diamine,Di-oleyl-sebacyl-tobramycin and Di-oleyl-dithioglycolyl-tobramycin. Eachpossibility is a separate embodiment.

In some exemplary embodiments, the cationic lipid may be selectedfrom:monocationic lipid N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammoniumpropane (DOTAP), BCAT O-(2R-1,2-di-O-(1′Z,9′Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate, BGSC(Bis-guanidinium-spermidine-cholesterol), BGTC(Bis-guanidinium-tren-cholesterol), CDAN (N′-cholesteryl oxycarbony1-3,7-diazanonane-1,9-diamine), CHDTAEA (Cholesterylhemidithiodiglycolyl tris(amino(ethyl)amine), DCAT(O-(1,2-di-O-(9′Z-octadecanyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate),DC-Chol (3β [N—(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol),DLKD (O,O′-Dilauryl N-lysylaspartate), DMKD (O,O′-DimyristylN-lysylaspartate), DOG (Diolcylglycerol, DOGS(Dioctadecylamidoglycylspermine), DOGSDSO(1,2-Dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine),DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine, DOSN (Dioleyl succinylethylthioneomycin), DOSP (Dioleyl succinyl paromomycin), DOST (Dioleylsuccinyl tobramycin), 1,2-Uiolcoyl-3-trimethyl ammoniopropane, DOTMA(N′[1-(2,3-Dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DPPES(Di-palmitoyl phosphatidyl ethanolamidospermine), DDAB and DODAP. Eachpossibility is a separate embodiment.

In some exemplary embodiments, the cationic lipid may be selected from:DLinDMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLin-MC3-DMA(heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), andDLin-KC2-DMA (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). Eachpossibility is a separate embodiment.

In some exemplary embodiments, the cationic lipid has a pKa in the rangeof about 6.5-7. In some embodiments, the cationic lipid is selectedfrom, but not limited to: DLinDMA, (with lipid pKa of 6.8), DLin-MC3-DMA(with lipid pKa of 6.44) and DLin-KC2-DMA (with lipid pKa of 6.7), orcombinations thereof. Each possibility is a separate embodiment.

In some embodiments, the membrane stabilizing lipids may be selectedfrom, but not limited to: cholesterol, phospholipids (such as, forexample, phosphatidylcholine (PC, such as, DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DMPC, DPPC, DHPC andDLPC), phosphatidylethanolamine, phosphatidylinositol,phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerols),cephalins, sphingolipids (sphingomyelins and glycosphingolipids),glycoglycerolipids, and combinations thereof. Each possibility is aseparate embodiment.

In some embodiments, the Phosphatidylethanolamines may be selected from,but not limited to: 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE),1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE)1,3-Dipalmitoyl-sn-glycero-2-phosphoethanolamine (1,3-DPPE),1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE),Biotin-Phosphatidylethanolamine,1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),Dipalmitoylphosphatidylethanolamine (DPPE),1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or combinationsthereof. In some embodiments, the Phosphatidylethanolamines may beconjugated to a PEG-Amine derivative. Each possibility is a separateembodiment.

According to some embodiments, the particles (lipid phase thereof), mayfurther include one or more PEG derivatives. In some embodiments, thePEG derivatives may be conjugated to one or more additional molecules,such as, a lipid. In some embodiments, the PEG derivative is selectedfrom, but not limited to: PEG-DMG, cDMA 3-N-(-methoxy poly(ethyleneglycol)2000)carbamoyl-1,2-dimyristyloxy-propylamine; PEG-cDSA,3-N-(-methoxy poly(ethyleneglycol)2000)carbamoyl-1,2-distearyloxy-propylamine, DSPE-PEG,PEG-maleimide, DSPE-PEG-maleimide, or combinations thereof. Eachpossibility is a separate embodiment.

In some embodiments, the maleimide derivative/moiety may be conjugated,attached or linked to a PEG-derivative, which may be by itselfconjugated, linked and/or attached to a lipid.

According to some embodiments, the ratio between the various lipids inthe particle may vary. In some embodiments, the ratio is a molar ratio.In some embodiments, the ratio is a weight ratio. In some embodiments,each of the lipid groups may be at molar ratio/a weight ratio of about1%-99%.

According to some embodiments, the weight ratio between the nucleic acidand the lipid mixture may be adjusted so as to achieve maximalbiological effect by the nucleic acid on the target site. In someembodiments, the ratio between the nucleic acid and the lipid phase maybe 1:1. For example, the weight ratio between the nucleic acid and thelipid phase may be 1:2. For example, the weight ratio between thenucleic acid and the lipid phase may be 1:5. For example, the weightratio between the nucleic acid and the lipid phase may be 1:10. Forexample, the weight ratio between the nucleic acid and the lipids phasemay be 1:16. For example, the weight ratio between the nucleic acid andthe lipid phase may be 1:20. In some embodiments, the weight ratiobetween the nucleic acid and the lipid phase is about 1:1 to 1:30 (w:w).

In some embodiments, the particles are nanoparticles. In someembodiments, the particles (including the nucleic acid encapsulatedwithin) and the targeting moiety on the surface particles have aparticle size (diameter) in the range of about 10 to about 500 nm. Insome embodiments, the particles have a particle size (diameter) in therange of about 10 to about 350 nm. In some embodiments, the particleshave a particle size (diameter) in the range of about 50 to about 250nm. In some embodiments, the particles have a particle size (diameter)in the range of about 10 to about 200 nm. In some embodiments, theparticles have a particle size (diameter) in the range of about 20 toabout 200 nm. In some embodiments, the particles have a particle size(diameter) in the range of about 50 to about 200 nm. In someembodiments, the particles have a particle size (diameter) in the rangeof about 70 to about 140 nm. In some embodiments, the particles have aparticle size (diameter) in the range of about 75 to about 200 nm. Insome embodiments, the particles have a particle size (diameter) in therange of about 90 to about 200 nm. In some embodiments, the particleshave a particle size (diameter) in the range of about 100 to about 200nm. In some embodiments, the particles have a particle size (diameter)in the range of about 120 to about 200 nm. In some embodiments, theparticles have a particle size (diameter) in the range of about 150 toabout 200. In some embodiments, the particles have a particle size(diameter) in the range of about 50 to about 150 nm. In someembodiments, the particles have a particle size (diameter) in the rangeof over about 10 nm. In some embodiments, the particles have a particlesize (diameter) of over about 20 nm. In some embodiments, the particleshave a particle size (diameter) of over about 30 nm. In someembodiments, the particles have a particle size (diameter) of over about40 nm. In some embodiments, the particles have a particle size(diameter) of over about 50 nm. In some embodiments, the particles havea particle size (diameter) of over about 60 nm. In some embodiments, theparticles have a particle size (diameter) of over about 70 nm. In someembodiments, the particles have a particle size (diameter) of over about80 nm. In some embodiments, the particles have a particle size(diameter) of over about 90 nm. In some embodiments, the particles havea particle size (diameter) of over about 100 nm. In some embodiments,the particles have a particle size (diameter) of over about 200 nm. Insome embodiments, the particles have a particle size (diameter) of notmore than about 500 nm. In some embodiments, the particles (includingthe nucleic acid encapsulated within) have a particle size (diameter) inthe range of about 5 to about 200 nm. In some embodiments, the particles(including the nucleic acid encapsulated within) have a particle size(diameter) in the range of about 70 to about 140 nm. In someembodiments, the particles (including the nucleic acid encapsulatedwithin) have a particle size (diameter) in the range of about 50 toabout 60 nm. In some embodiments, the particles (including the nucleicacid encapsulated within) have a particle size (diameter) in the rangeof about 55 to about 58 nm. In some embodiments, the size is a hydrodynamic diameter.

According to exemplary embodiments, the particles may be comprised of acationic lipid (such as, for example, DLinDMA, DLinMC3-DMA orDlinKC2-DMA), cholesterol, 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), PEG derivative (such as DMG-PEG) and PEG-maleimide conjugated toa lipid (such as DSPE-PEG-maleimide); at various mol:mol ratios, andfurther conjugated to a targeting moiety, wherein the targeting moietyis conjugated, linked, attached to the maleimide moiety. For example,the lipid phase may be comprised of:DLinMC3/DSPC/cholesterol/DMG-PEG/DSPE-PEG-Maleimide (mol/mol50:10:38:1.5:0.5). For example, the lipid phase may be comprised of:DLinMC3-DMA/Chol/DSPC/DMG-PEG/DSPE-PEG-maleimide (mol/mol50:38:10:1.95:0.05). For example, the lipid phase may be comprised of:DLinKC2-DMA/Chol/DSPC/DMG-PEG/DSPE-PEG-maleimide (mol/mol50:38:10:1.95:0.05). For example, the lipid phase may be comprised of:DLinKC2-DMA/Chol/DSPC/DMG-PEG/DSPE-PEG-maleimide (mol/mol45:30:23:1.5:0.5).

According to some embodiments, the lipid phase may comprise about 30-60%(mol:mol) cationic lipids. For example, the cationic lipid(s) maycomprise about 40-50% (mol:mol) of the lipid phase.

According to some embodiments, the lipid phase may comprise about 20-70%(mol:mol) membrane stabilizing lipids. For example, the membranestabilizing lipids may comprise about 40-60% of the lipid phase. In someembodiments, more than one type of membrane stabilizing lipid may beused in the lipid phase. For example, the membrane stabilizing lipid mayinclude cholesterol (being about 30-50% (mol:mol) of the lipid phase),and a phospholipid (such as, for example, DSPC), that may be about 5-15%(mol:mol) of the lipid phase.

According to some embodiments, the lipid phase may comprise about0.01-3% (mol:mol) of PEG-maleimide (optionally conjugated to a lipid).For example, the PEG-maleimide may comprise about 0.05-0.6% of the lipidmixture.

According to some embodiments, an additional PEG-derivative (conjugatedto a lipid) may comprise about 0.5-10% of the lipid phase composition.For example, the additional PEG derivative may comprise about 1.5-3% ofthe lipid phase.

According to some embodiments, there is provided a method for thepreparation of targeted particle(s) for delivery of a nucleic acid toleukocytes, the method comprising one or more of the steps of:

-   -   a) mixing a plurality of lipids, including, cationic lipid,        membrane stabilizing lipid and PEG-maleimide conjugated to a        phospholipid, in an organic solvent at a desired ratio;    -   b) adding nucleic acids to the mixture in a suitable solution at        a desired ratio;    -   c) mixing the lipid mixture and the nucleic acids in a        microfluidic micromixer to form particles;    -   d) dialyzing the particles to remove undesired solvents;    -   e) incubating the particles with a suitable targeting moiety to        generate targeted particles;    -   f) removing unconjugated/un-bound targeting moieties, optionally        by gel filtration;    -   g) filtration of reconstituted t-conjugated particles        encapsulating nucleic acid molecules;

According to some exemplary embodiments, there is provided a method forthe preparation of targeted particle(s) for delivery of a nucleic acidto leukocytes, the method comprising one or more of the steps of:

-   -   a) mixing a plurality of lipids, including, cationic lipid,        membrane stabilizing lipid and PEG-maleimide conjugated to a        phospholipid, in an organic solvent at a desired ratio;    -   b) adding nucleic acids to the mixture in a suitable solution at        a desired ratio;    -   c) mixing the lipid mixture and the nucleic acids in a        microfluidic micromixer to form particles;    -   d) dialyzing the particles to remove undesired solvents;    -   e) incubating the particles with reduced targeting antibodies to        generate targeted particles;    -   f) removing unconjugated antibodies, optionally by gel        filtration;    -   g) filtration of reconstituted t-conjugated particles        encapsulating nucleic acid molecules;

In some embodiments, the lipids are suspended in an acidic aqueousbuffer, such as, ethanol. In some embodiments, the nucleic acid is in anacetate buffer solution.

In some embodiments, the nucleic acid may be mixed with the lipidmixture in a microfluidizer mixer to form particlesencapsulating/carrying the nucleic acid.

In some embodiments, the targeting moiety may be manipulated prior tointeracting with the lipid particles, for example, by reduction. In someexemplary embodiments, when the targeting moiety is an antibody, itreduced by a reducing agent, such as, DTT. In some embodiments, thetargeting antibody is incubated with the particles after it has beenreduced. In some embodiments, the thiolized targeting antibody isincubated with the particles to form a conjugate with the maleimidemoiety.

According to some embodiments, the method for the preparation of thetargeted particles may include various modifications to finely adjustthe components of the composition, as well as the ratio between thecomponents, so as to obtain the most effective composition. Themodifications may include, for example, such parameters as, but notlimited to: the specific lipids used for the formation of the lipidcomposition, the ratio between the lipids of the lipid compositions, theidentity of the nucleic acid to be encapsulated, the ratio between thenucleic acid and the lipid composition, the specific targeting moietyused, the pH at which reactions are performed, the temperatures at whichreactions are performed, the conditions at which the reactions areformed, the time length of various steps, and the like, or anycombination thereof.

According to some embodiments, the method for the preparation of theparticles of the present invention may beneficially result in uniformlydistributed particle size.

According to some embodiments, the particles formed by the methods ofthe present invention may be lyophilized or dehydrated at various stagesof formation.

According to some embodiments, the targeted particles of the presentdisclosure can be used in the treatment of various leukocytes-associatedpathological conditions.

According to some embodiments, the particles may be administered as is.In some embodiments, the particles may be administered in a solution. Insome embodiments, the particles may be formulated to a suitablepharmaceutical composition to be administered by any desired route ofadministration. Exemplary routes of administration include such routesas, but not limited to: topical, oral or parenteral. Depending on theintended mode of administration, the compositions used may be in theform of solid, semi-solid or liquid dosage forms, such, as for example,tablets, suppositories, pills, capsules, powders, liquids, suspensions,or the like, preferably in unit dosage forms suitable for singleadministration of precise dosages. The pharmaceutical compositions mayinclude the cationic particles, a pharmaceutical acceptable excipient,and, optionally, may include other medicinal agents, pharmaceuticalagents, carriers, adjuvants, and the like. It is preferred that thepharmaceutically acceptable carrier be one which is inert to the nucleicacid encapsulated within the particles and which has no detrimental sideeffects or toxicity under the conditions of use. In some embodiments,the administration is localized. In some embodiments, the administrationis systemic.

In some embodiments, injectable formulations for parenteraladministration can be prepared as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol or the like. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, such as for example, sodiumacetate, sorbitan monolaurate, triethanolamine oleate, and the like.Aqueous injection suspensions may also contain substances that increasethe viscosity of the suspension, including, for example, sodiumcarboxymethylcellulose, sorbitol, and/or dextran. Optionally, thesuspension may also contain stabilizers. The parenteral formulations canbe present in unit dose or multiple dose sealed containers, such asampules and vials, and can be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier,such as, for example, water, for injections immediately prior to use. Insome embodiments, parenteral administration includes intravenousadministration.

In other embodiments, for oral administration, a pharmaceuticallyacceptable, non-toxic composition may be formed by the incorporation ofany of the normally employed excipients, such as, for example, mannitol,lactose, starch, magnesium stearate, sodium saccharine, talcum,cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesiumcarbonate, and the like. Such compositions include solutions,suspensions, tablets, dispersible tablets, pills, capsules, powders,sustained release formulations and the like. Formulations suitable fororal administration can consist of liquid solutions such as effectiveamounts of the compound(s) dissolved in diluents such as water, saline,or orange juice; sachets, lozenges, and troches, each containing apredetermined amount of the active ingredient as solids or granules;powders, suspensions in an appropriate liquid; and suitable emulsions.Liquid formulations may include diluents such as water and alcohols,(such as, for example ethanol, benzyl alcohol, and the polyethylenealcohols), either with or without the addition of a pharmaceuticallyacceptable surfactant, suspending agents, or emulsifying agents.

In determining the dosages of the particles to be administered, thedosage and frequency of administration may be selected in relation tothe pharmacological properties of the specific nucleic acidsencapsulated within the particles.

In some embodiments, there is provided a composition, which include aplurality of particles, wherein the various particles may encapsulate asimilar or different nucleic acid molecule (such as a similar ordifferent type of molecule, a similar or different sequence of thenucleic acid, and the like, or combinations thereof).

In some exemplary embodiments, particles comprising a nucleic acid, suchas, for example, siRNA, miRNA, shRNA, anti-sense RNA, and the like, maybe used in the treatment of various leukocyte-associated conditions,depending on the identity of the nucleic acid, the specific targetleukocyte, and the like. In some embodiments, the nucleic acidencapsulated within the particles may be a nucleic acid capable ofinducing silencing of a target gene. In some embodiments, the targetgene may be any gene, the expression of which is related to thecondition to be treated. In some embodiments, the target gene may be agene selected from, but not limited to: growth factors (such as EGFR,PDGFR), genes related to angiogenesis pathways (such as VEGF,Integrins), genes involved in intracellular signaling pathways and cellcycle regulation (such as PI3K/AKT/mTOR, Ras/Raf/MAPK, PDK1, CHK1, PLK1,Cyclins, STAT1, STAT3, MCL1, CKAP5, RRM1, SF3A1 and CDK11B). In someembodiments, a combination of nucleic acids, each having one or moretargets may be encapsulated within the particles. Each possibility is aseparate embodiment.

According to some embodiments, exemplary leukocyte-associated conditionsthat may be treated by the targeted particles may be selected from, butnot limited to: various types of cancer, various infections (such as,for example, viral infection, bacterial infection, fungal infection, andthe like), autoimmune diseases, neurodegenerative diseases,inflammations, and the like.

In some exemplary embodiments, the targeted particles comprising anucleic acid (such as, siRNA or miRNA, shRNA, anti-sense RNA, modifiedmRNA, guided RNA, or the like), may be used for the treatment of cancer.

In some embodiments, cancer is a disorder in which a population of cellshas become, in varying degrees, unresponsive to the control mechanismsthat normally govern proliferation and differentiation. In someembodiments, the cancer is a blood cancer. Non-limiting examples ofblood cancers are lymphoma, leukemia and multiple myeloma. Lymphomas maybe divided into two categories: Hodgkin lymphoma and non-Hodgkinlymphoma. Most non-Hodgkin lymphomas are B-cell lymphomas, that growquickly (high-grade) or slowly (low-grade). There are about 14 types ofB-cell non-Hodgkin lymphomas, including, but not limited to: Burkittlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma,marginal-zone B-cell lymphoma, Nodal marginal zone B cell lymphoma(NMZL), Splenic marginal zone lymphoma (SMZL), Intravascular largeB-cell lymphoma, Primary effusion lymphoma, Lymphomatoid granulomatosis,Primary central nervous system lymphoma, ALK-positive large B-celllymphoma, Plasmablastic lymphoma and mantle cell lymphoma (MCL). Eachpossibility is a separate embodiment. T-cell lymphomas include suchcancers as, but not limited to: Peripheral T-cell lymphoma, Anaplasticlarge cell lymphoma, Angioimmunoblastic Lymphoma, Cutaneous T-celllymphoma, Adult T-cell Leukemia/Lymphoma (ATLL), Blastic NK-cellLymphoma, Enteropathy-type T-cell lymphoma, Hematosplenic gamma-deltaT-cell Lymphoma, Lymphoblastic Lymphoma, Nasal NK/T-cell Lymphomas,Treatment-related T-cell lymphomas, and the like. Each possibility is aseparate embodiment.

In some exemplary embodiments, the nucleic acid that may be used for thetreatment of cancer are directed against a target gene, which isinvolved in the regulation of cell cycle. In some exemplary embodiments,the target gene may be Polo-like Kinase 1 (PLK), Cyclin D1, CHK1, Notchpathway genes, PDGFRA, EGFRvIII, PD-L1, RelB, STAT1, STAT3, MCL1, CKAP5,RRM1, SF3A1 and CDK11B, and the like, or combinations thereof. Eachpossibility is a separate embodiment.

According to some embodiments the particles disclosed herein areparticularly useful to specifically target and silence gene expressionin leukocytes cells, such as, B-cells and T-cells. In some embodiments,the particles may be administered systemically.

In some exemplary embodiments, the leukocytes cells are CD4+ T cells. Asexemplified herein, the targeted NLPs can effectively silence anexemplary gene, CD45, in CD4+ T cells at much lower doses (1 mg/Kg body)than any non-targeted system to leukocytes. In some embodiments, thissilencing is detected in all the major tissues that harbor T cells(blood, spleen, bone marrow and inguinal lymph nodes). In someembodiments, CD45 silencing was restricted to the CD4+ T cells and wasnot observed in other lymphocyte subsets. In some embodiments, thedecrease in CD45 expression is at the protein and/or mRNA levels.

According to some embodiments, there is a CD4+ T cell subset in whichuptake of the tLNPs was less efficient. According to some embodiments,and as exemplified herein two distinct CD4+ T cells populations thatdiffer in their tLNPs uptake ability can be identified. Distinctpopulation of CD4 T cells (CD4low) was permissive for tLNPsinternalization followed by siRNA cytoplasmic diffusion, while theremaining of the CD4+ cells harbor tLNPs (CD4high) on their surfacewithout proceeding to internalization.

According to some embodiments, tLNPs internalization and not endosomeescape is a central event that define tLNPs efficacy. According to someembodiments, the internalization of the tLNPs take place within a shorttime (for example, in the range of 30-120 minutes) post systemicadministration of the particles.

According to some embodiments, there is provided a method for thetreatment of leukocyte-related cancer, comprising the step ofadministration to a subject in need thereof the targeted particles ofthe present disclosure or a pharmaceutical compositions comprising thesame. In some embodiments, there is provided the use of the particles ofthe present disclosure or a pharmaceutical composition comprising thesame, for the treatment of leukocyte-related cancer. In someembodiments, the particles encapsulate a nucleic acid capable ofinducing growth inhibition or killing of the cancer cells, therebytreating the leukocyte-related cancer.

According to some embodiments and as exemplified herein, Mantle CellLymphoma (MCL) is used herein as a prototypic blood cancer fordemonstrating the targeting and therapeutic abilities of the particlesdisclosed herein. MCL is an aggressive B-cell lymphoma thatoverexpresses cyclin D1 with relatively poor prognosis. Thus,down-regulation of cyclin D1 using RNA interference (RNAi) can be usedas a therapeutic approach to this malignancy. According to someembodiments, and as exemplified herein, targeted lipid-basednanoparticles (LNPs) coated with anti-CD38 monoclonal antibodies arespecifically targeted to and taken up by human MCL cells in the bonemarrow of xenografted mice. According to some embodiments, when carryingsiRNAs against cyclin D1, CD38-targeted LNPs induce gene silencing inMCL cells to result in prolonged survival of tumor-bearing mice with noobserved adverse effects.

According to some embodiments, there is provided a method of treatingMCL, by administering an effective amount of the targeted particles ofthe present invention (or a pharmaceutical composition comprising thesame), whereby the targeted lipid based particles are coated(conjugated) to an anti-CD38 antibody (such as a monoclonal antibody).In some embodiments, the particles encapsulate/carry a nucleic acidmolecule capable of killing or inhibit growth of the MCL cells. In someembodiments, the nucleic acid molecule encapsulated within the particlesis an siRNA. In some embodiments, the siRNA is directed against a cellcycle regulator. In some embodiments, the siRNA is directed againstcyclin D1.

According to some embodiments, there is provided a use of the targetedparticles of the present invention for treating MCL, the particles (or apharmaceutical composition comprising the same) are coated/conjugated toan anti-CD38 antibody, such as, an anti-CD38 monoclonal antibody. Insome embodiments, the targeted particles carry/encapsulate an siRNAmolecule directed against a cell cycle regulator, such as, Cyclin D1.

According to some embodiments, and as exemplified herein, CD38 is asuitable target for antibody-mediated delivery of therapeutic siRNAs toMCL. LNPs-siRNA coated with an anti-CD38 monoclonal antibody (αCD38 mAb)exhibit specific MCL binding in vitro (in MCL cell lines and MCL primarylymphomas) and in vivo (in mice xenografted with a human MCL cell line).CD38-targeted LNPs (αCD38-LNPs) entrapping siRNA against cycD1 (siCycD1)are specifically taken up by MCL xenografts. αCD38-LNPs-siCycD1 inducedgene silencing and suppressed tumor cell growth in vitro, and prolongedthe survival of MCL-bearing mice.

According to some embodiments, αCD38-LNPs-siRNA can be used for treatingMCL and other CD38-expressing hematological malignancies. According tosome embodiments, there is provided a method of treating CD38-expressinghematological malignancies, the method comprising administering theαCD38-LNPs-siRNAs or pharmaceutical compositions comprising the same toa subject in need thereof.

According to some embodiments, αCD38-LNPs-siRNA that encapsulate siRNAsagainst cycD1 can prolong the survival subjects afflicted with MCL.

According to some embodiments, to achieve specificity for targeting MCLcells, theLNPs can be coated with an anti-CD38 mAb (such as, cloneTHB-7). In some embodiments, such mAb recognizes the surface proteinCD38, which is found on immature leukocyte precursors, but overexpressedin MCL tumor cells and other B-cell hematological malignancies, such asin CLL (where it correlates with poor prognosis) and multiple myeloma.According to some embodiments, the THB-7 αCD38 mAbs can be used as aMCL-targeting moiety without displaying significant anti-tumor activityby themselves. In some embodiments, an anti-CD38 antibody which displaysan anti-tumor effect by itself, may be used as a targeting moiety forthe particles, in which case, a synergistic effect on the targeted cellsmay be observed. In some embodiments, an anti-CD38 antibody can mediateactive delivery of siRNAs into the cytoplasm of targeted cells.

In some embodiments, αCD38-LNPs-siRNA may be internalized and inducegene silencing in various CD38-implicated diseases.

In some embodiments the αCD38-antibody is a THB-7 monoclonal antibody.

According to some embodiments, systemic administration of targetedlipid-based nanoparticles coated with antibodies targeting the CD38 cellmarker can specifically target (bind) MCL cells and induce an inhibitoryeffect within the cells, by the inhibitory siRNA encapsulated within theparticles. In some embodiments, the particles can provide protection invivo and in-vitro) to the nucleic acid molecules encapsulated therein.

According to some embodiments, the disclosed targeted particles exhibita safety profile, as repeated systemic administration did not affectbody weight.

According to some embodiments, the targeted particles disclosed hereinare particularly useful for MCL cells, other lymphomas and mosthematopoietic cells, which are dispersed throughout the body and are noteasily transfected by conventional lipid-based transfection reagents.The targeted particles disclosed are scalable, safe, efficient and allowhigh selectivity to subsets of leukocyte cells, in-vivo.

According to some embodiments, matching of the appropriate targetingmoiety to the surface receptor expressed on the target cells can beperformed to determine the targeting specificity of the particles. Forexample, it is possible to use different antibodies as targetingmoieties for MCL cells and other B cell malignancies (including thosethat do not express the CD38 protein). In some embodiments, whenmatching the appropriate targeting moiety to the target cell,consideration may be taken as some receptors might cluster on the cellsurface and induce an outside-in signaling event that could lead toproliferation, rather to inhibition of cellular growth.

According to some embodiments, the particles disclosed herein may beused as a diagnostic platform or a research tool for using in vivo geneknockdown to study leukocyte (such as, B cells and T-cells) biology.

In some embodiments, when treating a related condition, administrationof the targeted particles carrying a nucleic acid may be performed incombination with one or more additional treatments. For example, whentreating cancer, such combination therapy may be used to increase tumorsusceptibility to chemotherapy and irradiation.

In some embodiments, for treating cancer, silencing nucleic acids (suchas, siRNA, miRNA, shRNA, and the like) that target genes such as, butnot limited to: Cyclins, MGMT, Cx43, HeRI/EGF-R46, VEGF44, BCL-2, STAT1,STAT3, MCL1, CKAP5, RRM1, SF3A1, CDK11B Toll-like receptors, and thelike, may be used and may further provide synergistic responses.

In some embodiments, when treating a condition, repeated administrationof the targeted particles may be performed, wherein the dosagesadministered and the composition of the nucleic acid encapsulatedtherein may be identical, similar or different. In some embodiments, theadministration may be prolong (such as over the course of 1-120 hours).

The term comprising includes the term consisting of.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced be interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES

Materials and Methods:

Materials

-   Lipids: All lipids used for LNPs production (Cholesterol, DSPC and    DSPE PEG-Mal) were purchased from Avanti Polar lipids (USA).    Dlin-MC3-DMA was synthesized according to Cohen et.al.-   Monoclonal antibodies: FITC anti-mouse CD8 (clone 5H10-1), PerCP    anti-mouse CD3 (clone 145-2C11), PE anti-mouse CD19 (clone 6D5) and    PE anti-mouse CD4 (clone GK1.5) were purchased from BioLegend.    Anti-mouse CD4 (clone YTS.177) and Rat IgG2a isotype (clone 2A3)    were purchased from bioxcell. monoclonal antibodies THB-7 (mouse    IgG1 anti-hCD38) and MOPC-21 (mouse IgG1 isotype ctrl) were    purchased from BioXcell (USA).-   Secondary antibody Alexa Flour® 647 AffinityPure F(ab′)2 fragment    Donkey anti-rat IgG (H+L) (minimal cross-reaction to Mouse) was    purchased from Jackson ImmunoResearch Laboratories.-   siRNA molecules were designed and screened by Alnylam    Pharmaceuticals (USA).-   Chemically modified siRNAs sequences:    CD45 siRNA:

Sense strand: (SEQ ID NO: 1) cuGGcuGAAuuucAGAGcAdTsdT Anti-Sense strand: (SEQ ID NO: 2) UGCUCUGAAAUUcAGCcAGdTsdT Luc siRNA (siLuc):

Sense strand: (SEQ ID NO: 3) cuuAcGcuGAGuAcuucGAdTsdT Anti-Sense strand: (SEQ ID NO: 4) UCGAAGuACUcAGCGuAAGdTsdT 

-   Alexa-647-labeled siRNA possessed the same sequence as siLuc.    Cyclin D1 siRNA (siCycD1)

Sense strand (SEQ ID NO: 5) GUAGGACUCUCAUUCGGGATT 

-   2′-OMe modified nucleotides are in lower case, and phosphorothioate    linkages are represented by “s”.-   Cells: Granta-519 and Jeko-1 cells were purchased from DSMZ    (Germany) and TK-1, Mino and Rec-1 cells were purchased from the    American Type Culture Collection (ATCC) and cultured as recommended.    Preparation of Lipid-Based Nanoparticles (LNPs) Entrapping siRNAs

LNPs were prepared by using microfluidic micro mixture (Precision NanoSystems, Vancouver, BC, Canada) as described by Cohen et.al. One volumeof lipid mixtures (Dlin-MC3-DMA, DSPC, Chol, DMG-PEG and DSPE-PEG Mal at50:10:38:1.5:0.5 mole ratio or Dlin-MC3-DMA, Chol, DSPC, DMG-PEG,DSPE-PEG-Mal at 50:38:10:1.95:0.05 molar ratio, 9.64 nM total lipidconcentration) in ethanol and three volumes of siRNA (1:16 w/w siRNA tolipid) containing acetate buffer solutions were mixed by using dualsyringe pump (Model 5200, kD Scientific, Holliston, Mass.) to drive thesolutions through the micro mixer at a combined flow rate of 2 mL/minute(0.5 mL/min for ethanol and 1.5 mL/min for aqueous buffer). For labeledLNPs, 10% of Alexa-647 labeled siRNA were incorporated. For Cy5 labeledparticles, 10% Cy5 labeled non-targeted siRNA was used. The resultantmixture was dialyzed against PBS (pH 7.4) for 16 h to remove ethanol.

Conjugation of Targeting Antibodies with LNPs (tLNPS)

CD4 IgG (clone YTS 177) or Isotype mAbs (clone X63) were reduced with 1mM DTT for 30 min at room temperature. THB-7, isotype mAbs (MOPC-21)were reduced with DTT (1 mM and 5 mM, respectively) for 30 minutesshaking in 37° C. DTT was removed by using 7K cut off Zeba spindesalting columns (Thermo, USA) according to manufacturer protocol.

The thiolized mAbs were incubated with the Maleimide functionalized LNPsfor 1-2 h in gentle shaking at room temperature and optionally overnightat 4° C.

Removal of unconjugated mAbs was performed by loading the LNPs on gelfiltration chromatography columns containing sepharose CL-4B or CL-6Bbeads (Sigma-Aldrich, USA) with phosphate buffer saline (PBS) as amobile phase. The beads-column was washed with 0.1M NaOH and readjustedwith PBS prior to sample loading. The mAbs-LNPs-siRNA werereconcentrated via 10 k or 100 k Amicon Ultra-4 tubes (Millipore, USA)and optionally filtered through a 0.2 μm membrane (Sartorius, Germany).

Size, ζ-Potential and Ultrastructure Analysis of αCD38-LNPs-siRNA

LNPs size distribution and potential were determined by dynamic lightscattering using a Malvern nano ZS ζ-sizer (Malvern instruments, UK).For size measurements, LNPs were diluted 1:20 in PBS. All utilizedsamples showed a polydispersity index (PDI) lower than 0.2. For ζpotential measurements, LNPs were diluted 1:200 in DDW. Size and shapeof LNPs were analyzed by transmission electron microscopy (TEM). LNPs inPBS were placed on a formvar/carbon coated copper grid, air-dried andstained with 2% aqueous uranyl acetate. The analysis was performed witha Philips Tecnai F20 field emission TEM operated at 200 kV (USA). Insome cases, as indicated, size and zeta potential measurements wereperformed in water.

Transmission Electron Microscopy (TEM) Analysis

A drop of aqueous solution containing LNPs (with or without mAbs) wereplaced on the carbon coated copper grid and dried. The morphology ofLNPs was analyzed by Joel 1200 EX (Japan) transmission electronmicroscopy.

Confocal Microscopy Analysis:

One hour post administration of tLNPs, splenocytes were collected asmentioned above and stained with Hoechst (nucleus) and Calcein(Cytoplasm) labeling followed by anti-CD4-PE for membrane staining.Cells were washed and images were taken by Nikon C2 (Nikon Instrumentsinc., USA) confocal microscopy.

Dot Blot Analysis

Several concentration of Rat anti-CD4 (clone YTS.177) along with LNPs,tLNPs and IsoLNPs were blotted on nitrocellulose membrane. Afterblocking in 5% low-fat milk, the membrane was incubated with AffiniPureF(ab′)₂ Fragment ANTI-RAT conjugated to Horseradish Peroxidase (JacksonimmunoResearch) for 30 min in RT. ECL (Thermo Scientific Pierce) wasused as a substrate solution.

sCell Sorting and qPCR

Five days after tLNPs (siCD45) were injected and splenocytes wereisolated and stained with anti-CD4 PE and anti-CD45 AF647. As a control,mock treated splenocytes were stained with anti-CD4 PE. CD4⁺, CD45^(low)population from tLNP treated mice was collected CD4⁺ cells werecollected from mock mice as a control with FACSARIA (BD). mRNA wasisolated using EZ-RNA (Biological industries, Israel) and cDNA wasprepared using cDNA Synthesis kit (quanta biosciences) mouse PPIB wasused as endogenous control. Primers sequence: mPPIB FW: 5′ CCA TCG TGTCAT CAA GGA CTT C 3′ (SEQ ID NO: 6); mPPIB Rev: 5′ GAT GCT CTT TCC TCCTGT GCC 3′ (SEQ ID NO: 7); mCD45 FW: 5′ TCT TAC ACC ATC CAC TCT GGG C 3′(SEQ ID NO: 8); mCD45 Rev: 5′ GCT TCG TTG TGG TAG CTA TGG TT 3′ ((SEQ IDNO: 9).

Quantitative Real-Time PCR

Total RNA was isolated using EZ-RNA kit (Biological Industries, Israel)and cDNA was generated with qScript™ cDNA Synthesis Kit (Quanta, MD,USA) according to the manufacturers' instructions. qRT-PCR was performedwith Fast SYBR® Green Master Mix and the ABI StepOnePlus™ instrument(Life Technologies). Expression of cyclins was normalized to the two“house-keeping” genes eIF3a & eIF3c using the multiple endogenouscontrols option. This option allows the software to treat all endogenouscontrols as a single population, and calculates theexperiment-appropriate mean to establish a single value against whichthe target of interest is normalized. The primers used for amplificationare listed below (5′ to 3′):

CCND1 F: (SEQ ID NO: 10) GAGGAGCCCCAACAACTTCC; R: (SEQ ID NO: 11)GTCCGGGTCACACTTGATCAC. CCND2 F: (SEQ ID NO: 12) CGCAAGCATGCTCAGACCTT; R:(SEQ ID NO: 13) TGCGATCATCGACGGTGG. CCND3 F: (SEQ ID NO: 14)CTGACCATCGAAAAACTGTGCAT; R: (SEQ ID NO: 15) CCTCCCAGTCCCGCAA. eIF3a F:(SEQ ID NO: 16) TCCAGAGAGCCAGTCCATGC; R: (SEQ ID NO: 17)CCTGCCACAATTCCATGCT. eIF3c F: (SEQ ID NO: 18) ACCAAGAGAGTTGTCCGCAGTG; R:(SEQ ID NO: 19) TCATGGCATTACGGATGGTCC.Electroporation

-   1 nmole of each the siRNA duplexes (siLuc or siCycD1) were    electroporated into 10×10⁶ Granta-519 or Jeko-1 cells using the    Amaxa 4D-nuclefactor system (CM-119 program, SF solution).    In Vitro Cellular Uptake

Freshly isolated spelnocytes were incubated for 30 min at 4° C. withtLNPs (siCy5) followed by washing with PBS and incubation for 30 min at37° C. to allow internalization. After, cells were stained with anti-CD4PE and anti CD8-FITC. Cells were then analyzed by Nikon confocalmicroscope.

In Vivo tLNPs Biodistribution

Mice were intravenously injected with tLNPs (siCy5) at 1 mg/kg siRNA perbody weight of mouse. After one hour mice were sacrificed to collectblood, spleen, lymph nodes and bone marrow cells.

Isolation of Lymphocytes: Blood were collected in heparin coatedcollection tubes and the leukocytes isolated by density centrifugationusing ficoll paque plus (GE healthcare). Single cell suspensions ofsplenocytes were prepared by mincing of spleens and passing through a 70μm cell strainer (BD bioscience). RBCs were lysed using ACK lysis bufferand the resulting cells were resuspended in PBS. Inguinal lymph nodeswere isolated and minced to make single cell suspension. Cells werewashed twice with PBS followed by passing through a 70 um cell strainer.Cells were stained with a secondary Alexa 647 conjugated anti-Rat Fcantibody at 4° C. for 30 min; tLNPs were detected on the surface of thecells by Alexa 647-anti-Rat Fc and/or by Cy5. Cells were then washedwith PBS containing 1% FBS and incubated with labeled anti-CD4, CD8, andCD3 antibodies for 30 min at 4° C. Cells were washed and analyzed on aBecton Dickinson FACScalibur flow cytometer with CellQuest software(Becton Dickinson, Franklin Lakes, N.J.). Data analysis was performedusing FlowJo software (Tree Star, Inc., OR, USA). In addition, cellswere imaged on a Nikon confocal microscope.

In Vivo Silencing

8-6 weeks old C57BL6/J mice were obtained from the Animal BreedingCenter, Tel Aviv University (Tel Aviv, Israel). All animal protocolswere approved by the Tel Aviv Institutional Animal Care and UseCommittee. Mice were maintained and treated according NationalInstitutes of Health guidelines. tLNPs or isotype LNPs containing siRNAagainst CD45 or luciferase were injected intravenously (1 mg/kg siRNA).Mice were euthanized after 5 days and organs were collected for furtheranalysis.

In Vivo Immune Activation

LNPs were injected intravenously into C57BL mice. Lipopolysaccharide(LPS, Sigma) at a concentration of 1 mg/mL (100 μL) was used as apositive control. Blood was collected 2 hr after injection. Serum wasseparated and stored at −80° C. prior to cytokine analysis. Serumsamples were analyzed for cytokine levels according to manufacturerprotocol using the Milliplex® MAP kit (Millipore). The quantificationwas done based on standard curves for each cytokine.

In Vitro Binding Experiments

THB-7 (antiCD38) mAb was labeled with Alexa Fluor(R) 647 proteinlabeling kit (Invitrogen, USA). Binding of the labeled mAb to MCL celllines was assessed by flow cytometry (BD FACScalibur, USA, withCellQuest software for data collection and FlowJo software for dataanalysis). To determine the specific binding of αCD38-LNPs-siRNA, MCLcell lines expressing GFP and murine T-lymphoma TK1 cell line (0.5×10⁶each) were incubated together on ice with either 1% fetal calf serum PBSor with the buffer including 1 μg of free αCD38 mAb. After 15 minutes,αCD38-LNPs-siRNA with labeled siRNA were added (0.5 μg total siRNA) for30 additional minutes. Cells were collected for flow cytometry analysisafter 3 rounds of PBS wash. Cell populations were separately gated basedon GFP fluorescence.

In Vitro Internalization Experiments

0.5×10⁶ Granta cells were incubated in 50 μL of 1% serum PBS on 4degrees with alexa-647 αCD38 or isotype control mAbs for 10 minutes andthen incubated for 2 h at 37° C. (5% CO2). Then, cells were washedtwice, stained with PE-hCD20 mAbs (Biolegend, 302306) for 30 minutes onice, washed and subjected to confocal microscopy analysis. For assessingthe internalization of αCD38-LNPs-siRNA, 0.5×10⁶ Granta cells wereincubated in 50 μL of 1% serum PBS on ice with αCD38-, isotype- oruncoated siRNA-LNPs including labeled siRNA (500 ng of total siRNA).After 10 minutes, cells passed through 3 rounds of PBS wash and werere-incubated in fresh medium for 4 h at 37° C. (5% CO2). Then, cellswere washed, stained with PE-hCD20 mAbs for 30 minutes on ice, washedand subjected to confocal microscopy analysis. All pictures wereobtained on live cells using the Nikon Eclipse C2 configured with NI-Emicroscope and processed with NIS-elements software using X40 objectivemagnification (Nikon, Japan).

In Vitro Gene Silencing

0.3×10⁶ Granta-519 or Jeko-1 cells were placed in tissue culture24-wells plates with 0.5 mL of full medium. αCD38-LNPs-siCycD1 orαCD38-LNPs-siLuc were added to the wells (2 μg of siRNA for eachcondition). After 18 h incubation in standard culture conditions, cellswere washed 3 times and re-incubated in fresh medium in cultureconditions. 48 h following initial exposure to treatments, cells werecollected for cycD1 protein quantification, mRNA quantification or cellcycle measuring. cycD1 intracellular staining was performed according tothe BD Pharmingen™ Transcription Factor Buffer set instructions usingrabbit anti human cycD1 monoclonal antibody (Cell marque, 241R-16) orisotype control (Jackson ImmunoResearch, 011-000-003) at 0.68 μg/mL.Cells were washed and incubated with 2 μg/mL of Alexa647 Donkeyanti-rabbit antibody (Jackson ImmunoResearch, 711-605-152) for 30minutes at 4° C., washed twice and analysed by flow cytometry. Thegeometric mean of detected Alexa Fluor®-647 fluorescence intensity forat least 5000 cells was used as the compared value for each sample.cycD1 relative expression for each treatment group was derived from thequotient of the value of cycD1 staining divided by the value of isotypectrl staining.

Cell Cycle Studies

The transfected cells were washed with ice-cold PBS, and fixed with 70%ethanol for 1 h. Then, the cells were washed twice with cold PBS andincubated for 10 min at 37° C. in 250 μL PBS with 10 μg/mL propidiumiodide (PI), 2.5 μg/mL DNase-free RNase A (Sigma, USA) and 0.01%Triton-X. PI fluorescence was assessed by flow cytometry. Analyzes byFlowJo™ were performed on at least 9000 cells per samples after gatingout debris and cell duplets based on the FL2-Area/FL2-Width channels.Cell cycle distributions were obtained via the application of theDean-Jett-Fox model on gated cells with RMS scores ranging between 1.5and 2.5.

Ex Vivo Binding with Human MCL Primary Samples

Peripheral blood samples were obtained from MCL patients treated at theRabin medical center, (Petah Tikva, Israel) and the Chaim Sheba MedicalCenter at Tel Hashomer, (Ramat Gan, Israel) in accordance withinstitutional review board-approved informed consent. Mononuclear cellswere extracted from full blood samples using Ficoll-Paque™ PLUS (GEHealthcare, UK). 1×10⁶ of cells from the primary sample were incubatedwith targeted LNPs and free competing αCD38 mAb as described in the invitro binding experiments. After 3 rounds of wash, cells were stainedwith CD19 (Biolegend, 302219) and CD45 (Biolegend, 304008) mAbs for 30minutes on ice. Membranal staining was used during analysis to separateB-lymphocytes (CD19+/CD45+) from non-B leukocytes populations(CD19−/CD45+) while assessing for siRNA fluorescence.

Human MCL Xenograft Mouse Model

To enable easier identification of the MCL cells in vivo, Granta-519cells were stably infected with pTurbo-GFP retroviral particles (kindlysupplied by Prof. Eran Bacharach). The infected cells were sortedaccording to their GFP expression, and the highest GFP population(Granta-GFP) was collected and grew.

For modeling MCL in vivo, Female C.B-17/IcrHsd-Prkdcscid mice werepurchased from Harlan laboratories (Jerusalem, Israel). The mice werehoused and maintained in laminar flow cabinets under specificpathogen-free (SPF) conditions in the animal quarters of Tel AvivUniversity and in accordance with current regulations and standards ofthe Israel Ministry of Health. All animal protocols were approved by TelAviv University Institutional Animal Care and Use Committee.

2.5×10⁶ Granta-519 or Granta-GFP cells were intravenously injected into8 weeks old mice. Mice were monitored daily and killed when diseasesymptoms appeared (15% reduction in body weight or hind leg paralysis).Different tissues and organs (liver, kidney, lungs, spleen, blood, bonemarrow, and if existed, solid tumor) were collected with respect to thedifferent experiments, and processed into single cell suspensions. Toidentify the MCL cells, cell suspensions were analyzed by flowcytometry, using PE mouse anti human CD45 antibody, PE or alexa 647mouse anti human CD20 (Biolegend, 302318) antibodies and/or GFPexpression. Femour bones were fixed, sliced, decalcified and stainedwith H&E as described before (38).

In Vivo Binding of αCD38 mAb

MCL xenograft mice were intravenously injected with 30 μg of Alexa-647labeled αCD38 or isotype control mAbs at day of hind-leg paralysisappearance. 2 h later, mice were scarified and liver, kidneys, lungs,spleen and bone marrow were harvested and processed into single cellsuspensions. αCD38 fluorescence on cells was assessed by flow cytometry.

In Vivo Binding of Targeted LNPs

At day 24 post tumor injection, either saline, isotype- orαCD38-LNPs-siCycD1 including labeled siRNA (1.25 mg siRNA/kg body) wereadministered i.v. via the tail vein. After 2 h, mice were sacrificed andcells from the bone marrow extracted. Single cell suspensions wereprepared by passing the cells through 70 μm cell strainers (BD) andwashing with PBS. Cells were stained with Alexa Fluor®-488-hCD20(Biolegend, 302316) and PE-mCD45 (Biolegend 103106) mAbs for 30 minutesand washed prior to analysis. Human MCL cells (GFP+/hCD20+/mCD45−) andmurine cells (GFP−/hCD20−/mCD45+) were gated separately and assessed forsiRNA fluorescence. The cells from the mock-treated mouse were used as abaseline for negative fluorescence, while cells in other groups wereconsidered positive to siRNA fluorescence if exhibiting higher valuesthan the 99th percentile's value of mock.

Survival Experiment

The survival experiment was performed in Charles Rivers Laboratories,Tranent, Scotland, in accordance with local ethical requirements. 30 MCLxenograft mice were divided into 3 treatment groups: untreated (mock),αCD38-LNPs-siLuc and αCD38-LNPs-siCycD1. The different treatments (1 mgsiRNA/kg body) were injected via retro-orbital route 9 times (days 5, 8,12, 15, 19, 22, 26, 29, 33). Mice displaying loss of 15% bodyweight orlimb paralysis were euthanized.

Statistical Analysis

In experiments with multiple groups, one-way ANOVA with Bonferronicorrection was sued. For the comparison of two experimental groups,two-tailed Student's t test was used. Differences between or amonggroups are labeled as n.s. for not significant, * for p<0.05, ** forp<0.005 and *** for p<0.0005. A value of P<0.05 was consideredstatistically significant. Analyzes were performed with Prism 6(Graphpad Software).

Example 1: Preparation and Characterization of Targeted-LipidNanoparticles (tLNPs), Targeted to T-Cells

To prepare the effective siRNA delivery system for T cells, whichinclude siRNA-encapsulated LNPs a microfluidic mixer system(Nanoassemblr) was used. The particles were prepared as detailed above.The mixing of acidified siRNAs (pH 4) with a mixture of lipids(cholesterol, DSPC, PEG-DMG, Dlin-MC3-DMA and DSPE-PEG-maleimide;illustrated in FIG. 1), resulted in the production of highlyuniform-sized nanoparticles (NPs) that had a mean diameter of ˜58 nmmeasured by dynamic light scattering (DLS). Since the pKa ofDlin-MC3-DMA lipid is 6.44, it is expected to provide a minimal orneutral charge at physiological pH (7.4) and indeed the zeta potentialof the LNPs was shown to be about −9 mV (Table 1). The particleshydrodynamic diameter was also confirmed using transmission electronmicroscopy (TEM), as shown in FIG. 2 (left hand panel). To constructT-cells targeted LNPs (tLNPs), DTT reduced monoclonal antibodies (mAbs)against CD4 (clone YTS.177) were chemically conjugated to the maleimidefunctional group of the LNPs. This procedure resulted in tLNPs with amean diameter of ˜130 nm and a zeta potential of ˜−10 mV (Table 1). Thissize was also validated using TEM (FIG. 2, right hand panel) and theincrease in size of tLNPs could be explained by the chemical conjugationof the mAb followed by concentration of the LNPs using an Amiconcentrifugal filter. siRNA encapsulation efficiency was tested using aRibogreen assay. An encapsulation efficiency of close to 100%, wasdetermined, since no free siRNA was detected.

Antibody presence on the surface of LNPs was confirmed using a dot plotassay, as shown in FIG. 3.

TABLE 1 Characterization of LNPs by dynamic light scattering and Zetapotential measurements. Data are presented as mean ± SD of sixindependent measurements. LNPs CD4-tLNPs Hydro dynamic diameter (d · nm)58 ± 6 129 ± 5  PDI  0.1 ± 0.05  0.12 ± 0.02 Zeta potential (mV) −9.3 ±0.3 −10 ± 0.5 siRNA encapsulation (%) 95 ± 2 95 ± 9 

Example 2: Preparation and Characterization of Targeted-LipidNanoparticles (tLNPs), Targeted to B-Cells (MCL Cells)

Lipid-nanoparticles (LNPs) encapsulating siRNAs using a microfluidicmixing system as described were constructed (FIG. 1B). The lipid mixtureincluded the lipid Dlin-MC3-DMA (50 mol %), cholesterol (38%), DSPC(10%), DMG-PEG (1.95%) and DSPE-PEG-Maleimide (0.05%). αCD38 mAb (cloneTHB-7) was reduced to allow its chemical conjugation to maleimide groupspresent in the LNPs and then incubated with the LNPs. TheαCD38-LNPs-siRNA had a mean diameter of ˜116 nm with a narrow sizedistribution (PDI˜0.157) as measured by dynamic light scattering (DLS,Table 2). ζ-potential measurements showed a slight negative surfacecharge, as expected, at physiological pH (21). Transmission electronmicroscopy (TEM) analysis of the LNPs indicated a globular shape andsize distribution in accordance with the DLS measurements (FIG. 2B).

TABLE 2 Characterization of αCD38-LNPs-siRNA by dynamic light scatteringand ζ-potential measurements. Hydrodynamic diameter   116 ± 7.9 nmPolydispersity index 0.157 ± 0.017  ζ Potential −5.83 ± 1.1 mVData are represented as mean±s.d. out of 12 (size & PDI) or 2(ζ-potential) measurements for independently produced batches. Allindividual measurements included 3 technical replicates.

Example 3: Specific Binding of tLNPs to CD4⁺ T Cells Ex Vivo

For the siRNA to be delivered to the cell, the targeting moiety shouldinduce internalization of the nucleic acid encapsulated within theparticles. To test whether tLNPs can mediate specific targeting of siRNAto CD4⁺ cells and induce internalization, tLNPs or LNPs coated with anisotype control mAb (isoLNPs) encapsulating Cy5 labeled-siRNA (siCy5)were used to treat a heterogeneous population of primary C57BL/6splenocytes ex vivo. To measure binding to the target cells, tLNPs(siCy5) or isoLNPs (siCy5) were incubated with heterogeneous populationof splenocytes, and analyzed by flow cytometry. As shown in FIG. 4A,significant and robust binding to CD4⁺ cells was observed for tLNPs((orange) dots; right upper quartet) treated mice, while no significantbinding was observed for isoLNPs treated mice (grey dots; upper leftquartet). Non-specific binding to either CD8⁺ cells or B cells (leftlower quartet) for mice treated tLNPs/isoLNPs was observed (FIG. 4A).Representative bar graphs are shown in FIG. 4B demonstrating thatsignificant amount (˜100%) of CD4⁺ cells bind tLNPs, as compared toisoLNPs (˜1%).

To test the tLNPs uptake, an additional stage of incubation at 37° C.for 30 min was added following tLNPs binding. Confocal microscopy wasused to demonstrate the uptake of tLNPs into the target cells. As shownin FIG. 4C, the uptake of tLNPs exclusively into CD4⁺ cells based onco-labeling of the siCy5 containing cells with anti-CD4-PE (formembrane), calcein and Hoechst (for cytoplasm and nuclei staining) tovalidate tLNPs internalization. tLNPs specificity was also validated bylabeling CD8⁺ T cell subset, that unlike CD4⁺, did not demonstrate tLNPsuptake (Supporting information FIG. S2). Hence, anti-CD4 mAb coated LNPsare suitable to deliver siRNAs into CD4⁺ cells.

Example 4: Targeted Silencing in Blood Circulating CD4⁺ T Cells

To examine the capability of tLNP delivering siRNAs to silence geneexpression in CD4⁺ T cells in vivo, tLNPs containing siRNAs against CD45(tLNPs (siCD45)) were prepared. CD45, a cell surface tyrosinephosphatase, was chosen since it is a pan-leukocyte marker and thus canbe used for testing specific silencing in different leukocyte subsets.First, it was tested whether tLNPs can target circulating CD4⁺ T cells.Mice were intravenously injected with tLNPs (siCy5) or isoLNPs (siCy5)as control. One hour post administration, blood was harvested, stainedfor markers of different leukocytes subsets and analyzed by flowcytometry. The results shown in FIG. 5A demonstrate that, remarkably,all CD4⁺ cells bound tLNPs, while none of the other leukocytes subsetsexamined showed Cy5 labeling compared to isoLNPs or mock treated controlgroups. Representative bar graphs are shown in FIG. 5B.

After confirming in vivo binding, the ability of tLNPs (siCD45) toknockdown CD45 expression in C57BL/6 mice was tested. To this end,several LNPs were used as controls, including tLNPs that wereconstructed using siRNA against luciferase (tLNPs(siluc)) and isoLNPs(siCD45) or LNPs (siCD45) (unmodified, non-conjugated LNPs). Five dayspost administration, blood was harvested and cells were stained forCD45. As shown in FIG. 5C, only mice treated with tLNPs (siCD45)resulted in CD45 silencing in leukocytes compared to the control groups.Representative histograms showing a significant reduction of CD45 levelsin mice treated with tLNPs compared to isoLNPs and other control groupsare presented in FIG. 5D. The data presented was performed intriplicates and reproduced with two independent experiments and with twodifferent productions of LNPs. To test the specificity of silencing, thecells harvested from the treated animals were stained with anti-CD3PerCP and anti-CD4 PE antibodies (FIG. 5E). Remarkably, almost 100% ofthe silenced cells were CD4⁺ T cells. Accordingly, a very highspecificity using the tLNPs in circulating CD4⁺ T cells was obtained.

Next, the tLNPs were tested for immune toxicity. To this aim, a panel ofcytokines, including, TNF-α, IL-17 and IL-10 were tested, in micetreated with either the tLNPs (siCD45) or LPS, a potent TLR activator,as a positive control. As shown in FIG. 6, only a mild elevation ofcytokines was observed in tLNPs (siCD45) treated mice over untreatedmice.

Example 5: tLNPs Induce Gene Silencing in CD4⁺ T Cells in DifferentHematopoietic Organs

One of the challenges associated with leukocyte targeted therapies isthat in order to manipulate all leukocytes of a specific subset, such asCD4⁺ T cells, the delivery agent needs to reach diverse organs withinthe body, since leukocyte are distributed across multiple tissues,including spleen, lymph nodes and bone marrow. To determine the breadthof siRNA delivery across lymphoid tissues, a systematic examination ofthe binding of tLNPs to CD4⁺ T cells in different hematopoietic organswas performed. Mice were injected intravenously with tLNPs (siCy5) orisoLNPs (siCy5). One hour post injection, cells from the spleen,inguinal lymph nodes and bone marrow were isolated and stained with aset of antibodies to detect different leukocytes subsets (anti-CD3,anti-CD4 and anti-CD8). Remarkably, all CD4⁺ T cells from each of thetissues tested showed specific binding of the tLNPs, while no binding toany of the organs has observed in mice treated with isoLNPs (FIG. 7).Although all the tissues showed binding to the CD4⁺ T cells, the bindinglevel in different organs was heterogeneous.

As shown in FIG. 8A, the highest binding was observed in bloodcirculating CD4⁺ T cells, while the lowest binding was observed in lymphnodes. This heterogeneity could result from different tLNPs kineticsacross different tissues. After demonstrating the selective binding ofthe tLNPs to CD4⁺ T cells in all the tested organs, its ability toinduce potent gene silencing was examined. Five days post i.v.administration of tLNPs (siCD45), cells from blood, spleen, lymph nodesand bone marrow were isolated and co-stained for CD45, CD3, CD8 and CD4expression. Flow cytometry analysis of the CD3⁺, CD4⁺ cell populationsclearly shows specific CD45 knockdown from each of these analyzedtissues (FIG. 8B). As shown in FIG. 8C, the most effective silencing wasobserved in CD4⁺ T cells of lymph nodes with 36% of the cells stainingnegative for CD45 compared to the mock treated cells followed by theperipheral blood (35%), spleen (31%) and bone marrow (24%). The resultspresented in FIG. 8D show the silencing of CD45 in CD4⁺ T cells at themRNA level. Notably, other cell types showed no decrease in their CD45expression levels (FIG. 9).

To examine whether tLNPs amount is a limiting factor, the binding andsilencing efficacy of tLNPs at higher tLNPs doses (2 mg/kg siRNA) wastested. As shown in FIG. 10A, a significant higher specific binding inblood and bone marrow (FIG. 10A) of the mice treated with 2 mg/kg siRNAcompared with the dose frequently used (1 mg/kg) is observed. Afunctional experiment using higher dose of tLNPs (siCD45) did not resultin higher silencing. As shown in FIG. 10B, a dose response experimentusing 0.5, 1 and 2 mg/kg siRNA, showed a significant advantage to 1mg/kg compare with 0.5 mg/kg in the lymph nodes, without any significantchange in silencing with 2 mg/kg dose. To further validate siRNAsilencing, CD4⁺ T cells, CD45^(KD) (KD-knock down) cells from the spleenof the tLNPs treated animals and CD4⁺ cells from mock treated animalswere isolated by using Cell sorter (BD FACSAriaIII™). CD45 mRNAexpression levels were analyzed by quantitative real time PCR (qRT-PCR).As shown in FIG. 8D, a significant (˜80%) decrease in CD45 mRNA levelswas observed in CD4⁺ T cells collected from tLNPs treated mice comparedwith mock treated CD4⁺ T cells.

Example 6: tLNPs are Internalized by Distinct CD4 Subset Followed byFunctional Silencing

After establishing tLNPs as a platform strategy to specifically silencegenes of interest using siRNAs in circulating and resting CD4+ Tlymphocytes (using the pan leukocyte surface marker CD45 as a genemodel), the mechanism that underlie the differences between the two CD4⁺T cell populations that differ in their response to the tLNPs wasinvenstigated. One demonstrate CD45 silencing and the other do not alterits CD45 expression levels. Intriguingly, two distinct CD4⁺ T cellpopulations were noticed in all tissues tested in binding experimentsafter 1 h (FIG. 8A) or 4 h after tLNPs administration. One shows highCD4 PE staining, as in mock cells (CD4^(high)) and the other presentslow CD4 PE staining (CD4^(low)). CD4^(low) population represents CD4⁺ Tcells and not a different population of cells since this population ofcells stained positively for the T cell co-receptor CD3 encompassingboth the CD4^(high) and CD4^(low) cell populations (FIGS. 11A-B). ThisCD4^(low) population of cells was not found in the untreated mice. Sincethe percent of the CD4^(low) population resembled the silencing percentobtained in the silencing assays, it was tested whether the reduction inanti-CD4 PE can be a result of CD4 receptor sequestering from themembrane due to tLNPs internalization, followed by silencing. Todetermine if there was a correlation between low CD4 surface expressionand tLNP internalization, the level of internalized tLNPs (siCy5) wastested by both flow cytometry and confocal microscopy analysis. For flowcytometer assay a secondary antibody (anti-Rat Fc) directed against thefragment crystallizable (Fc) region of the CD4 mAb of tLNPs was used.This experimental method is not designed to detect internalizingparticles but rather cell surface bound particles. Flow cytometryanalysis clearly demonstrates that although the amount of siCy5 issimilar between CD4^(low) and CD4^(high) cells, CD4^(low) population hasless tLNPs on the surface, since CD4^(low) have reduced staining ofanti-Rat Fc compared to CD4^(high) population (FIG. 12A). These resultswere validated by confocal microscopy, in which cytoplasm, nuclei andCD4 membrane were stained with calcein, Hoechst and anti-CD4respectively to insure tLNPs cytoplasmic localization. As shown in FIG.12B, CD4^(low) cells have effectively internalized the tLNPs (siCy5), onthe other hand, CD4^(high) cells had lower levels of internalized tLNPs(siCy5) with the majority of tLNPs located on the surface (FIG. 12B).Therefore, these results demonstrate that two populations of CD4⁺ cells(CD4^(low) and CD4^(high) cells) in the tLNP treated samples may reflectthe degree of internalization and sequestration of the CD4 molecules.This could suggest that there may be differences between CD4⁺ cellspopulations in their ability to endocytose the tLNPs and may explain theCD45 knockdown efficiency as seen in FIG. 8B.

Next, it was aimed to confirm that CD4^(low) cells not only internalizethe tLNPs, but also that this internalization leads to silencing. Thiswill determine that the bottleneck for gene silencing in CD4 cells usingtLNPs system is the internalization step. For this purpose, anexperiment in which CD4^(low) and CD4^(high) populations are separatedby FACS Sorter (FIG. 12C), one hour after administration of tLNPs(siCD45) in vivo and CD45 expression levels are tested after 3 days inculture. As a control, CD4⁺ cells from mice treated with iso LNPs(siCD45), in which no CD4^(low) population was observed were sorted.Remarkably, up to 70% silencing was observed in CD4^(low) populationscompared with 16% in CD4^(high) population. Neglected 2% silencing wasobserved in CD4^(high) population of isoLNPs treated group (FIG. 12E).

Revealing that CD4^(low) is the silenced population also contributes todecipher how lymph node CD4^(+ T cells) exhibit high knockdown efficacywhile showing the lowest tLNPs uptake. Following uptake of the particlesat the single cell level is limited when using siCy5 due to degradationof the florescence dye along with florescence decay due to low endosomalpH. Indeed Cy5 labeling is dramatically decrease among all tissues 4 hpost administration (FIG. 13A), however since CD4^(low) represent thesilenced cells that internalize the tLNPs, their fate may be followed.Interestingly, after 4 hours a significant increase of ˜5.5% inCD4^(low) in lymph nodes accompanied by a trend of a CD4^(low) decreasein other tissues can be detected (FIG. 13B). This might imply that moretLNPs accumulate in the lymph after 4 hours or that CD4 cells havemigrated to the lymph.

Example 7: Model Establishment—MCL Cells are Mainly Engrafted in theBone Marrow of SCID Mice

To test the ability of αCD38-LNPs-siCycD1 to target dispersed MCL cells,an animal model of disseminated MCL in which MCL cells home to the bonemarrow (BM), as in the advanced stages of the human disease wasestablished. Granta-519 cells (2.5×10⁶) stably expressing GFP(Granta-GFP) were injected intravenously (i.v.) into 6-8 weeks oldfemale C-mB-17 SCID mice. These mice developed hind-leg paralysis after24 to 30 days, at which time liver, lungs, spleen, kidney, blood and BMcells were harvested to assess the distribution of MCL cells by flowcytometry. Granta-GFP cells consistently homed to the bone marrow (FIG.14A). There were also some tumor cells in the lung, but very few in theliver, kidney, spleen or circulation. Bone marrow tumors that displacednormal bone marrow were prominent in H&E stained femoral slices, asshown in FIG. 14B.

Example 8: CD38—a Receptor Target for MCL

Targeting siRNAs selectively to tumors requires the identification of acell surface receptor that is over-expressed on tumor cells compared tomost other tissues, whose binding leads to endocytosis and release ofendocytosed siRNAs into the target cell cytoplasm. Consistent withprevious reports (Chang et. al.), it was found that CD38 is highly andbroadly expressed on four tested MCL lines (FIG. 15A) and on humanprimary MCL samples (FIG. 15B). In vitro incubation of Granta-519 cellswith fluorescently labeled CD38 mAb (clone THB-7, αCD38) led tointernalization of the antibody-receptor complex (FIG. 15C). Next,labeled αCD38 mAb binding after i.v. injection into mice bearingGranta-GFP lymphomas was checked (FIG. 15D). Virtually all of the GFP+lymphoma cells in the BM and lung bound the antibody. While only fewnormal GFP− liver, blood or lung bound αCD38, about half of GFP− spleencells and a quarter of kidney cells bound it, but the staining wasgenerally less intense than for the GFP+ tumor cells. These findingsindicate the potential of THB-7 mAb and the CD38 receptor to serve astargeting moiety and target receptor, respectively, for specificdelivery of LNPs to MCL in vivo.

Example 9: αCD38-LNP-siRNA Specifically Bind and Internalize into MCLCells

To test whether αCD38-LNPs-siRNA specifically bind to MCL cells, humanMCL were co-coltured with CD38− (negative) mouse T lymphoma TK-1 celllines and the mixtures were with αCD38-LNPs-siRNA (as prepared inExample 2) containing fluorescently labeled siRNAs (FIG. 16A). siRNAuptake was determined by flow cytometry. αCD38-LNPs-siRNA selectivelybound to the MCL cell lines, as indicated by higher fluorescenceintensity levels in those cells. Moreover, addition of free unlabeledαCD38 mAbs to the co-cultures decreased particles' binding to backgroundlevels, indicating that binding was via CD38. Similar results wereobtained using two primary human MCL samples (FIG. 16B). Next, incubatedGranta-519 cells were incubated with LNPs that were uncoated or coatedwith αCD38 or an isotype control antibody and entrapped fluorescentlylabeled siRNAs. Following incubation, the cells were imaged by confocalmicroscopy using αCD20 to stain their cell surface (FIG. 16C). Bound andinternalized siRNA was only detected with the αCD38-coated LNPs.

Example 10: αCD38-LNPs-siCycD1 Induce Robust Gene Knockdown and CellCycle Arrest

Next it was examined whether αCD38-LNPs loaded with cycD1 siRNA(siCycD1), or as control luciferase siRNA (siLuc), could mediate genesilencing in two MCL cell lines, Granta-519 and Jeko-1 (FIG. 17A andFIG. 17B). When these MCL cell lines were treated withαCD38-LNPs-siCycD1 they exhibited an average 55.7% (P<0.001) and 56%(P<0.002) reduction in CycD1 protein levels determined by flow cytometrycompared to αCD38-LNPs-siLuc. The latter particles did not significantlyaffect CycD1 levels. CycD1 knockdown was also confirmed at the mRNAlevel by qRT-PCR (FIG. 17C). The reduction in CycD1 levels in theαCD38-LNPs-siCycD1 incubated cells caused a cell cycle arrest in theG₀/G₁ phase (FIG. 17D). This effect was evident even though downregulation of cycD1 induced compensatory elevation of other D cyclinsexpression. As shown in FIG. 17E, qRT-PCR analysis of CCND1, CCND2 andCCND3 mRNA levels 24, 48, 72 and 96 hours post electroporation inGranta-519 (left) and Jeko-1 (right) cells reveales that Cyclin D2 wasconsistently overexpressed following treatment with siCycD1. Cyclin D3exhibited lower or stable expression following treatment, before apronounced increase at Day 4 post electroporation. Expression wasnormalized to both eIF3a and eIF3c genes and depicted as mRNAconcentration relative to cells electroporated with siLuc.

CycD1 overexpression is a prominent genetic hallmark and tumorigenicfactor in MCL. The relevance of selective cycD1 silencing in MCL hasbeen questioned before due to compensatory elevation of cyclin D2expression. As shown, it was detected that the down-regulation of cycD1expression induces a compensatory upregulation of cyclin D2 expression(FIG. 17E). A similar compensatory expression pattern regarding cyclinD3 was detected as well. The compensatory expression of these genes didnot mask the effects of cycD1 silencing in MCL cells (as demonstrated inFIGS. 17B and 17D). Studies have shown that the compensatory activity ofother D cyclins allow cycD1 knockouts mice to be viable and to show onlylimited developmental defects. Therefore it is reasonable that undesirednon-specific uptake of αCD38-LNPs-siCycD1 by non-MCL bystander cellswould exhibit only low or no adverse effects.

Example 11: αCD38-Coated LNPs Specifically Target MCL Cells In Vivo

The ability of αCD38-LNP-siRNA to deliver siRNAs into Granta-519xenografts in vivo, was tested. When hind leg paralysis appeared,MCL-bearing mice were mock treated or treated i.v. with LNPs, loadedwith fluorescently labeled siRNAs, those were coated with αCD38 or anisotype control antibody. BM was extracted 2 hours later and analyzed byflow cytometry for siRNA uptake into mouse CD45+ cells and the humantumor, stained with anti-human CD20 antibody (FIG. 18A and FIG. 18B).Fluorescent siRNAs were detected in ˜30% of MCL cells in mice treatedwith αCD38-LNP-siRNA, compared to ˜6% of isotype-LNPs-siRNA (P<0.0002).Although about 15% of mouse BM cells were labeled with the fluorescentsiRNA, there was no significant difference in siRNA accumulation betweenmice treated with αCD38 or control antibody-coated LNPs (P=0.38). ThusαCD38-LNPs-siRNA specifically bind to MCL cells in the BM in vivo.

Example 12: CD38-LNPs-siCycD1 Induce Therapeutic Gene Silencing in MCLCells In Vivo

-   The therapeutic effect of CD38-LNPs-siCycD1 on the survival of    MCL-bearing mice was tested. Mice (n=10/group) were treated biweekly    with 9 i.v. injections of 1 mg/kg siRNA, starting 5 days after tumor    inoculation. Control mice were mock treated or treated with    CD38-LNPs-siLuc. No loss in body weight was observed during the    first 21 days of the experiment, indicating that the treatments did    not induce major adverse effects (FIG. 18D). Treatment with    αCD38-LNPs-siCycD1 increased median survival from 34 to 49 days    (P=0.0087) compared to αCD38-LNPs-siLuc treatment (FIG. 18C).    Survival of mice treated with the luc-targeting control LNPs was not    significantly different from survival of mock treated mice. These    findings demonstrate the beneficial therapeutic effect of using    siCycD1 in vivo in MCL-bearing mice.

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The invention claimed is:
 1. A particle for targeted delivery of anucleic acid to a leukocyte cell, the particle comprising: a lipidmembrane suitable for encapsulating a nucleic acid, wherein the lipidmembrane comprises Dlin-MC3-DMA, cholesterol, DSPC, DMG-PEG, andDSPE-PEG-maleimide conjugated to a targeting moiety and wherein thetargeting moiety is an antibody selected from the group consisting of ananti-CD38 antibody, an anti-CD4 antibody, an anti-CD8 antibody, and ananti-CD3 antibody, or an antigen binding fragment thereof.
 2. Theparticle of claim 1, further comprising a nucleic acid encapsulatedwithin the particle.
 3. The particle of claim 1, wherein the leukocytecells are primary lymphocytes.
 4. The particle of claim 3, wherein thelymphocytes are selected from B-cells and T-cells.
 5. The particle ofclaim 1, which is internalized by the leukocyte cell.
 6. The particle ofclaim 1, wherein the targeting moiety is a single antibody configured tospecifically recognize an antigen expressed by said leukocyte.
 7. Theparticle of claim 1, wherein the targeting moiety is an anti-CD38antibody.
 8. The particle of claim 1, wherein the targeting moiety is anantibody selected from an anti-CD4 antibody, an anti-CD8 antibody, andan anti-CD3 antibody.
 9. The particle of claim 2, wherein the nucleicacid is the only molecule having a biological effect on the target site.10. The particle of claim 2, wherein the percentage of encapsulation ofthe nucleic acid is over about 90%.
 11. The particle of claim 2, whereinthe nucleic acid comprises an interfering RNA selected from the groupconsisting of siRNA, miRNA, shRNA, and antisense RNA, modified formsthereof or combinations thereof.
 12. The particle of claim 11, whereinthe nucleic acid is an siRNA.
 13. The particle of claim 12, wherein thetargeting moiety is an anti-CD38 antibody.
 14. The particle of claim 2,wherein the nucleic acid comprises an siRNA against a cell cycleregulator selected from the group consisting of: Polo-like Kinase 1(PLK), Cyclin D1, CHK1, Notch pathway genes, PDGFRA, EGFRvIII, PD-L1,RelB, STAT1, STAT3, MCL1, CKAP5, RRM1, SF3A1 and CDK11B, or anycombinations thereof.
 15. The particle of claim 14, wherein the cellcycle regulator is Cyclin D1.
 16. A composition comprising a pluralityof particles according to claim
 1. 17. A composition comprising aplurality of particles according to claim 2.